Ketoreductase polypeptides for the synthesis of chiral compounds

ABSTRACT

The present disclosure provides engineered ketoreductase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase enzyme. Also provided are polynucleotides encoding the engineered ketoreductase enzymes, host cells capable of expressing the engineered ketoreductase enzymes, and methods of using the engineered ketoreductase enzymes to synthesize a variety of chiral compounds.

The present application claims priority to U.S. patent application Ser.No. 15/543,535 filed Jul. 13, 2017, which is a national stageapplication filed under 35 USC § 371 and claims priority tointernational application to PCT International Application No.PCT/US2016/016692, filed Feb. 5, 2016, which claims priority to U.S.Prov. Pat. Appln. Ser. No. 62/114,380, filed Feb. 10, 2015, all of whichare hereby incorporated by reference, in their entireties and for allpurposes.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The Sequence Listing concurrently submitted herewith under 37 C.F.R. §1.821 in a computer readable form (CRF) via EFS-Web as file nameCX2-148WO1_ST25.txt is herein incorporated by reference. The electroniccopy of the Sequence Listing was created on Feb. 4, 2016, with a filesize of 456 Kbytes.

FIELD OF THE INVENTION

The present invention provides engineered ketoreductase enzymes havingimproved properties as compared to a naturally occurring wild-typeketoreductase enzyme, as well as polynucleotides encoding the engineeredketoreductase enzymes, host cells capable of expressing the engineeredketoreductase enzymes, and methods of using the engineered ketoreductaseenzymes to synthesize a variety of chiral compounds.

BACKGROUND

Enzymes belonging to the ketoreductase (KRED) or carbonyl reductaseclass (EC1.1.1.184) are useful for the synthesis of optically activealcohols from the corresponding prochiral ketone substrate and bystereoselective reduction of corresponding racemic aldehyde substrates.KREDs typically convert ketone and aldehyde substrates to thecorresponding alcohol product, but may also catalyze the reversereaction, oxidation of an alcohol substrate to the correspondingketone/aldehyde product. The reduction of ketones and aldehydes and theoxidation of alcohols by enzymes such as KRED requires a co-factor, mostcommonly reduced nicotinamide adenine dinucleotide (NADH) or reducednicotinamide adenine dinucleotide phosphate (NADPH), and nicotinamideadenine dinucleotide (NAD) or nicotinamide adenine dinucleotidephosphate (NADP) for the oxidation reaction. NADH and NADPH serve aselectron donors, while NAD and NADP serve as electron acceptors. It isfrequently observed that ketoreductases and alcohol dehydrogenasesaccept either the phosphorylated or the non-phosphorylated co-factor (inits oxidized and reduced state), but not both.

In order to circumvent many chemical synthetic procedures for theproduction of key compounds, ketoreductases are being increasinglyemployed for the enzymatic conversion of different keto and aldehydesubstrates to chiral alcohol products. These applications can employwhole cells expressing the ketoreductase for biocatalytic ketone andaldehyde reductions, or by use of purified enzymes in those instanceswhere presence of multiple ketoreductases in whole cells would adverselyaffect the stereopurity and yield of the desired product. For in vitroapplications, a co-factor (NADH or NADPH) regenerating enzyme such asglucose dehydrogenase (GDH), formate dehydrogenase etc. can be used inconjunction with the ketoreductase. It is desirable to identify otherketoreductase enzymes that can be used to carryout conversion of variousketo substrates to corresponding chiral alcohol products.

SUMMARY OF THE INVENTION

The present invention provides engineered ketoreductase enzymes havingimproved properties as compared to a naturally occurring wild-typeketoreductase enzyme, as well as polynucleotides encoding the engineeredketoreductase enzymes, host cells capable of expressing the engineeredketoreductase enzymes, and methods of using the engineered ketoreductaseenzymes to synthesize a variety of chiral compounds.

The present disclosure provides engineered ketoreductase (“KRED”)enzymes that are capable of stereoselectively reducing a defined ketosubstrate to its corresponding alcohol product and having an improvedproperty when compared with the naturally-occurring, wild-type KREDenzyme obtained from L. kefir (SEQ ID NO:2) or when compared with otherengineered ketoreductase enzymes.

Moreover, the engineered enzymes described herein can have one or moreimproved properties in addition to the altered stereoselectivity. Forexample, the engineered ketoreductase polypeptide can have increasedenzymatic activity as compared to the wild-type ketoreductase enzyme forreducing the substrate to the product and/or further increasesstereoselectivity for the (S) enantiomer. Improvements in enzymeproperties can also include, among others, increases in thermostability,solvent stability, and/or reduced product inhibition.

The present invention provides engineered polypeptides comprising aminoacid sequences with at least 80% sequence identity to SEQ ID NO:2 and atleast one substitution at a position selected from X68, X94, X102, X110,X114, X135, X144, X145, X147, X149, X150, X153, X158, X173, X175, X190,X196, X197, X198, X199, X201, X202, X203, X205, X206, X207, X209, X210,X211, X212, X213, X217, X233, X249, X250, and X252, as compared to SEQID NO:2, and wherein the polypeptide has greater 2a:2c selectivity ascompared to SEQ ID NO:2. In some embodiments, the amino acid sequencesof the engineered polypeptides comprise at least one of the following:the residue corresponding to X68 is a non-polar, or aliphatic residue;the residue corresponding to X94 is a polar, or non-polar residue; theresidue corresponding to X102 is an acidic residue; the residuecorresponding to X110 is an acidic, or aromatic residue; the residuecorresponding to X114 is a non-polar residue; the residue correspondingto X135 is a basic residue; the residue corresponding to X144 is anon-polar, or aromatic residue; the residue corresponding to X145 is anaromatic residue; the residue corresponding to X147 is a non-polar, oraliphatic residue; the residue corresponding to X149 is a polar residue;the residue corresponding to X150 is an aromatic, or acidic residue; theresidue corresponding to X153 is a polar, or aromatic residue; theresidue corresponding to X158 is a non-polar, or aliphatic residue; theresidue corresponding to X173 is a non-polar, or aliphatic residue; theresidue corresponding to X175 is a polar residue; the residuecorresponding to X190 is an aromatic, aliphatic, non-polar, or polarresidue; the residue corresponding to X196 is an aromatic, polar, basic,non-polar, or aliphatic residue; the residue corresponding to X197 is anaromatic residue; the residue corresponding to X198 is a non-polar,polar residue; the residue corresponding to X199 is an acidic residue;the residue corresponding to X201 is a polar residue; the residuecorresponding to X202 is an aromatic, or basic residue; the residuecorresponding to X203 is an aromatic, non-polar, or aliphatic residue;the residue corresponding to X205 is a polar residue; the residuecorresponding to X206 is an aromatic residue; the residue correspondingto X207 is an aromatic, acidic, non-polar, or aliphatic residue; theresidue corresponding to X209 is an aromatic, acidic, non-polar, polar,or aliphatic residue; the residue corresponding to X210 is a non-polar,or aliphatic residue; the residue corresponding to X211 is an aromatic,acidic, aliphatic, polar, or non-polar residue; the residuecorresponding to X212 is an aromatic, basic, non-polar, aliphatic, oracidic residue; the residue corresponding to X213 is an aromatic, basic,acidic, polar, non-polar, or aliphatic residue; the residuecorresponding to X217 is a polar, or aromatic residue; the residuecorresponding to X233 is a polar residue; the residue corresponding toX249 is a basic, or non-polar residue; the residue corresponding to X250is a non-polar residue; and the residue corresponding to X252 is anaromatic residue, as compared to SEQ ID NO:2. In some additionalembodiments, the amino acid sequences of the engineered polypeptidescomprise at least one substitution selected from X68V, X94G/M/Q, X102D,X110E/W, X114G, X135K, X144G/W, X145W, X147L, X149S, X150D/F/P/W,X153H/P/T/V/C, X158V, X173L, X175T, X190A/L/Q/T/W, X196H/I/K/N/Q, X1961,X196K/N/Q, X197Y, X198G/S, X199E, X201N, X202F/H/R/Y, X203G/L/W, X205T,X206H/W/Y, X207D/G/V/Y, X209E/F/G/I/M/T/V/W, X210L/P, X211E/H/I/P/S,X212A/E/G/H/N/P/R/V, X213D/E/G/H/K/M/N/R, X217H/N, X233Q/T, X249G/K/R,X250G, and X252W, as compared to SEQ ID NO:2. In some furtherembodiments, the amino acid sequences of the engineered polypeptidescomprise a substitution at position X190, as compared to SEQ ID NO:2. Instill some additional embodiments, the amino acid sequences of theengineered polypeptides comprise a substitution at position X190,wherein the substitution is selected from P, A, T, and Q, as compared toSEQ ID NO:2. In some embodiments, the amino acid sequences of theengineered polypeptides comprise a substitution at position X249, ascompared to SEQ ID NO:2. In some additional embodiments, the amino acidsequences of the engineered polypeptides comprise a substitution atposition X249, wherein the substitution is selected from R, K, and G, ascompared to SEQ ID NO:2. In some further embodiments, the amino acidsequences of the engineered polypeptides comprise substitutions atpositions X190 and X249, as compared to SEQ ID NO:2. In still somefurther embodiments, the amino acid sequences of the engineeredpolypeptides comprise substitutions at positions X190 and X249, whereinthe substitution at position X190 is selected from P, A, T, and Q, andthe substitution at position 249 is selected from R, K, and G, ascompared to SEQ ID NO:2.

The present invention also provides engineered polypeptides comprisingamino acid sequences with at least 80% sequence identity to SEQ ID NO:4,and at least one substitution at a position selected from X94, X144,X145, X150, X190, and X249, as compared to SEQ ID NO: \4, and whereinsaid polypeptide has greater 2a:2c selectivity as compared to SEQ IDNO:4. In some embodiments, the amino acid sequences of the engineeredpolypeptides comprise at least one of the following: the residuecorresponding to X145 is an aromatic residue; the residue correspondingto X249 is a basic residue; the residue corresponding to X94 is anon-polar residue; the residue corresponding to X144 is a non-polarresidue; the residue corresponding to X150 is a residue; and the residuecorresponding to X190 is an aromatic residue, as compared to SEQ IDNO:4. In some additional embodiments, the amino acid sequences of theengineered polypeptides comprise at least one substitution selected fromX145W, X249K/R, X94G, X144G, X150P, X190W, as compared to SEQ ID NO:4.

The present invention also provides engineered polypeptides comprisingamino acid sequences with at least 80% sequence identity to SEQ ID NO:10and at least one substitution at a position selected from X145, X150,X153, X190, X198, X206, and X211, as compared to SEQ ID NO:10, andwherein said polypeptide has greater 2a:2c selectivity as compared toSEQ ID NO:10. In some embodiments, the amino acid sequences of theengineered polypeptides comprise at least one of the following: theresidue corresponding to X145 is an aromatic residue; the residuecorresponding to X150 is an aromatic residue; the residue correspondingto X153 is a residue; the residue corresponding to X190 is an aliphaticor non-polar residue; the residue corresponding to X198 is a non-polarresidue; the residue corresponding to X206 is an aromatic residue; andthe residue corresponding to X211 is an aromatic, acidic, aliphatic,polar, or non-polar residue, as compared to SEQ ID NO:10. In somefurther embodiments, the amino acid sequences of the engineeredpolypeptides comprise at least one substitution selected from X145W,X150W/F, X153C, X190A, X198G, X206Y, and X211I/H/S/E, as compared to SEQID NO:10.

The present invention also provides engineered polypeptides comprisingamino acid sequences with at least 80% sequence identity to SEQ ID NO:26and at least one substitution at a position selected from X94, X147,X153, X175, X190, X196, X199, X201, X202, X203, X206, X207, X209, X210,X211, X212, X213, X217, as compared to SEQ ID NO:26, and wherein saidpolypeptide has greater 2a:2c selectivity as compared to SEQ ID NO:26.In some embodiments, the amino acid sequences of the engineeredpolypeptides comprise at least one of the following: the residuecorresponding to X147 is a non-polar, or aliphatic residue; the residuecorresponding to X153 is a polar, or aromatic residue; the residuecorresponding to X175 is a polar residue; the residue corresponding toX190 is a polar, aliphatic, or non-polar residue; the residuecorresponding to X196 is a polar, basic, non-polar, or aliphaticresidue; the residue corresponding to X199 is an acidic residue; theresidue corresponding to X201 is a polar residue; the residuecorresponding to X202 is an aromatic, or basic residue; the residuecorresponding to X203 is an aromatic, non-polar, or aliphatic residue;the residue corresponding to X206 is an aromatic residue; the residuecorresponding to X207 is an aromatic, acidic, or non-polar residue; theresidue corresponding to X209 is an aromatic, acidic, non-polar, polar,or aliphatic residue; the residue corresponding to X210 is a non-polar,or aliphatic residue; the residue corresponding to X211 is a non-polarresidue; the residue corresponding to X212 is an aromatic, basic,non-polar, or acidic residue; the residue corresponding to X213 is anaromatic, basic, acidic, polar, non-polar, or aliphatic residue; theresidue corresponding to X217 is a polar residue; and the residuecorresponding to X94 is a polar, or non-polar residue, as compared toSEQ ID NO:26. In some further embodiments, the amino acid sequences ofthe engineered polypeptides comprise at least one substitution selectedfrom X94Q/M, X147L, X153T/H, X175T, X190Q/L, X196N/K/I, X199E, X201N,X202Y/R/H/F, X203W/L, X206W/H, X207Y/G/D, X209Y/W/V/T/M/I/G/F/E,X210P/L, X211P, X212R/H/G/E, X213T/R/N/M/K/H/G/E/D, and X217N, ascompared to SEQ ID NO:26.

The present invention also provides engineered polypeptides comprisingamino acid sequences with at least 80% sequence identity to SEQ ID NO:42and at least one substitution at a position selected from X68, X94,X102, X110, X114, X135, X149, X150, X153, X158, X173, X190, X196, X197,X198, X203, X205, X207, X209, X212, X213, X217, X233, X249, X250, X252,as compared to SEQ ID NO:42, and wherein said polypeptide has greater2a:2c selectivity as compared to SEQ ID NO:42. In some embodiments, theamino acid sequences of the engineered polypeptides comprise at leastone of the following: the residue corresponding to X102 is an acidicresidue; the residue corresponding to X110 is an acidic, or aromaticresidue; the residue corresponding to X114 is a non-polar residue; theresidue corresponding to X135 is a basic residue; the residuecorresponding to X149 is a polar residue; the residue corresponding toX150 is an acidic residue; the residue corresponding to X153 is aresidue; the residue corresponding to X158 is a non-polar, or aliphaticresidue; the residue corresponding to X173 is a non-polar, or aliphaticresidue; the residue corresponding to X190 is a non-polar, polar, oraliphatic residue; the residue corresponding to X196 is an aromatic, orpolar residue; the residue corresponding to X197 is an aromatic residue;the residue corresponding to X198 is a polar residue; the residuecorresponding to X203 is a non-polar, aromatic, aliphatic residue; theresidue corresponding to X205 is a polar residue; the residuecorresponding to X207 is a non-polar, or aliphatic residue; the residuecorresponding to X209 is a non-polar residue; the residue correspondingto X212 is a non-polar, aliphatic, acidic, or aromatic residue; theresidue corresponding to X213 is an acidic, basic, non-polar, aliphatic,or polar residue; the residue corresponding to X217 is an aromaticresidue; the residue corresponding to X233 is a polar residue; theresidue corresponding to X249 is a non-polar residue; the residuecorresponding to X250 is a non-polar residue; the residue correspondingto X252 is an aromatic residue; the residue corresponding to X68 is anon-polar, or aliphatic residue; and the residue corresponding to X94 isa polar residue. In some additional embodiments, the amino acidsequences of the engineered polypeptides comprise at least substitutionselected from X68V, X94Q, X102D, X110E/W, X114G, X135K, X149S, X150D,X153P, X158V, X173L, X190L/T, X196H/N/Q, X197Y, X198S, X203G/L/W, X205T,X207V, X209G, X212A/E/G/H/N/P, X213E/G/K/M/N/T/R/V, X217H, X233Q/T,X249G, X250G, and X252W, as compared to SEQ ID NO:42.

The present invention also provides engineered polypeptides, wherein thestereoselective activity of the polypeptides is increased at least2-fold as compared to the corresponding activity of the referencepolypeptide of SEQ ID NOS:4, 10, 26, and/or 42.

The present invention also provides engineered polypeptides in which theamino acid sequence comprises a sequence selected from the groupconsisting of the even-numbered sequence identifiers of SEQ IDNOS:6-238.

The present invention also provides engineered polypeptides havingketoreductase activity, wherein the polypeptide comprises SEQ ID NO:240.In some embodiments, the residue corresponding to X173 is leucine. Insome alternative embodiments, the residue corresponding to X249 isglycine. In some further embodiments, the residue corresponding to X173is leucine and the residue corresponding to X249 is glycine.

The present invention also provides engineered polypeptides havingketoreductase activity, wherein the polypeptide comprises SEQ ID NO:239.In some embodiments, the residue corresponding to X173 is leucine. Insome alternative embodiments, the residue corresponding to X249 isglycine. In some further embodiments, the residue corresponding to X173is leucine and the residue corresponding to X249 is glycine.

The present invention also provides polynucleotides encoding theengineered polypeptides provided herein. In some embodiments, thepolynucleotide comprises a nucleic acid sequence selected from the groupconsisting of the odd-numbered sequence identifiers of SEQ ID NOS:5-237.

The present invention also provides expression vectors comprising thepolynucleotide(s) provided herein.

The present invention also provides host cells comprising thepolynucleotide(s) provided herein or at least one expression vectorprovided herein.

The present invention also provides methods of preparing engineeredpolypeptides having ketoreductase activity, comprising culturing thehost cell provided herein, under conditions suitable for expression ofthe polypeptide, optionally further comprising isolating the engineeredpolypeptide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides the reaction scheme.

FIG. 2 provides the structures of substrate and product isomers.

DESCRIPTION OF THE INVENTION

The present invention provides engineered ketoreductase enzymes havingimproved properties as compared to a naturally occurring wild-typeketoreductase enzyme, as well as polynucleotides encoding the engineeredketoreductase enzymes, host cells capable of expressing the engineeredketoreductase enzymes, and methods of using the engineered ketoreductaseenzymes to synthesize a variety of chiral compounds.

Definitions

In reference to the present disclosure, the technical and scientificterms used in the descriptions herein will have the meanings commonlyunderstood by one of ordinary skill in the art, unless specificallydefined otherwise. Accordingly, the following terms are intended to havethe following meanings. All patents and publications, including allsequences disclosed within such patents and publications, referred toherein are expressly incorporated by reference. Unless otherwiseindicated, the practice of the present invention involves conventionaltechniques commonly used in molecular biology, fermentation,microbiology, and related fields, which are known to those of skill inthe art. Unless defined otherwise herein, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention, thepreferred methods and materials are described. Indeed, it is intendedthat the present invention not be limited to the particular methodology,protocols, and reagents described herein, as these may vary, dependingupon the context in which they are used. The headings provided hereinare not limitations of the various aspects or embodiments of the presentinvention.

Nonetheless, in order to facilitate understanding of the presentinvention, a number of terms are defined below. Numeric ranges areinclusive of the numbers defining the range. Thus, every numerical rangedisclosed herein is intended to encompass every narrower numerical rangethat falls within such broader numerical range, as if such narrowernumerical ranges were all expressly written herein. It is also intendedthat every maximum (or minimum) numerical limitation disclosed hereinincludes every lower (or higher) numerical limitation, as if such lower(or higher) numerical limitations were expressly written herein.

As used herein, the term “comprising” and its cognates are used in theirinclusive sense (i.e., equivalent to the term “including” and itscorresponding cognates).

As used herein and in the appended claims, the singular “a”, “an” and“the” include the plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to a “host cell” includes aplurality of such host cells.

Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation and amino acid sequences are written left to rightin amino to carboxy orientation, respectively.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention that can be had by reference to thespecification as a whole. Accordingly, the terms defined below are morefully defined by reference to the specification as a whole.

“Ketoreductase” and “KRED” are used interchangeably herein to refer to apolypeptide having an enzymatic capability of reducing a carbonyl groupto its corresponding alcohol. More specifically, the ketoreductasepolypeptides of the invention are capable of stereoselectively reducingthe compound of formula (I) to the corresponding product of formula(II), as shown in Scheme 1 (See, FIG. 1).

As used herein, the terms “protein,” “polypeptide,” and “peptide” areused interchangeably herein to denote a polymer of at least two aminoacids covalently linked by an amide bond, regardless of length orpost-translational modification (e.g., glycosylation, phosphorylation,lipidation, myristilation, ubiquitination, etc.). Included within thisdefinition are D- and L-amino acids, and mixtures of D- and L-aminoacids.

As used herein, “polynucleotide” and “nucleic acid’ refer to two or morenucleosides that are covalently linked together. The polynucleotide maybe wholly comprised ribonucleosides (i.e., an RNA), wholly comprised of2′ deoxyribonucleotides (i.e., a DNA) or mixtures of ribo- and 2′deoxyribonucleosides. While the nucleosides will typically be linkedtogether via standard phosphodiester linkages, the polynucleotides mayinclude one or more non-standard linkages. The polynucleotide may besingle-stranded or double-stranded, or may include both single-strandedregions and double-stranded regions. Moreover, while a polynucleotidewill typically be composed of the naturally occurring encodingnucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), itmay include one or more modified and/or synthetic nucleobases (e.g.,inosine, xanthine, hypoxanthine, etc.). Preferably, such modified orsynthetic nucleobases will be encoding nucleobases.

As used herein, “coding sequence” refers to that portion of a nucleicacid (e.g., a gene) that encodes an amino acid sequence of a protein.

As used herein, “naturally occurring” or “wild-type” refers to the formfound in nature. For example, a naturally occurring or wild-typepolypeptide or polynucleotide sequence is a sequence present in anorganism that can be isolated from a source in nature and which has notbeen intentionally modified by human manipulation.

As used herein, “non-naturally occurring” or “engineered” or“recombinant” when used in the present disclosure with reference to(e.g., a cell, nucleic acid, or polypeptide), refers to a material, or amaterial corresponding to the natural or native form of the material,that has been modified in a manner that would not otherwise exist innature, or is identical thereto but produced or derived from syntheticmaterials and/or by manipulation using recombinant techniques.Non-limiting examples include, among others, recombinant cellsexpressing genes that are not found within the native (non-recombinant)form of the cell or express native genes that are otherwise expressed ata different level.

As used herein, “percentage of sequence identity,” “percent identity,”and “percent identical” refer to comparisons between polynucleotidesequences or polypeptide sequences, and are determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence for optimal alignment of the two sequences. Thepercentage is calculated by determining the number of positions at whicheither the identical nucleic acid base or amino acid residue occurs inboth sequences or a nucleic acid base or amino acid residue is alignedwith a gap to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity. Determination of optimal alignment and percentsequence identity is performed using the BLAST and BLAST 2.0 algorithms(See e.g., Altschul et al., J. Mol. Biol. 215: 403-410 [1990]; andAltschul et al., Nucleic Acids Res. 3389-3402 [1977]). Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information website.

Briefly, the BLAST analyses involve first identifying high scoringsequence pairs (HSPs) by identifying short words of length Win the querysequence, which either match or satisfy some positive-valued thresholdscore T when aligned with a word of the same length in a databasesequence. T is referred to as, the neighborhood word score threshold(Altschul et al, supra). These initial neighborhood word hits act asseeds for initiating searches to find longer HSPs containing them. Theword hits are then extended in both directions along each sequence foras far as the cumulative alignment score can be increased. Cumulativescores are calculated using, for nucleotide sequences, the parameters M(reward score for a pair of matching residues; always >0) and N (penaltyscore for mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (See e.g., Henikoff and Henikoff, Proc Natl Acad Sci USA 89:10915[1989]).

Numerous other algorithms are available and known in the art thatfunction similarly to BLAST in providing percent identity for twosequences. Optimal alignment of sequences for comparison can beconducted using any suitable method known in the art (e.g., by the localhomology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 [1981];by the homology alignment algorithm of Needleman and Wunsch, J. Mol.Biol. 48:443 [1970]; by the search for similarity method of Pearson andLipman, Proc. Natl. Acad. Sci. USA 85:2444 [1988]; and/or bycomputerized implementations of these algorithms [GAP, BESTFIT, FASTA,and TFASTA in the GCG Wisconsin Software Package]), or by visualinspection, using methods commonly known in the art. Additionally,determination of sequence alignment and percent sequence identity canemploy the BESTFIT or GAP programs in the GCG Wisconsin Software package(Accelrys, Madison Wis.), using the default parameters provided.

As used herein, “reference sequence” refers to a defined sequence towhich another sequence is compared. A reference sequence may be a subsetof a larger sequence, for example, a segment of a full-length gene orpolypeptide sequence. Generally, a reference sequence is at least 20nucleotide or amino acid residues in length, at least 25 residues inlength, at least 50 residues in length, or the full length of thenucleic acid or polypeptide. Since two polynucleotides or polypeptidesmay each (1) comprise a sequence (i.e., a portion of the completesequence) that is similar between the two sequences, and (2) may furthercomprise a sequence that is divergent between the two sequences,sequence comparisons between two (or more) polynucleotides orpolypeptide are typically performed by comparing sequences of the twopolynucleotides over a comparison window to identify and compare localregions of sequence similarity. The term “reference sequence” is notintended to be limited to wild-type sequences, and can includeengineered or altered sequences. For example, in some embodiments, a“reference sequence” can be a previously engineered or altered aminoacid sequence.

As used herein, “comparison window” refers to a conceptual segment of atleast about 20 contiguous nucleotide positions or amino acids residueswherein a sequence may be compared to a reference sequence of at least20 contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

As used herein, “corresponding to”, “reference to” or “relative to” whenused in the context of the numbering of a given amino acid orpolynucleotide sequence refers to the numbering of the residues of aspecified reference sequence when the given amino acid or polynucleotidesequence is compared to the reference sequence. In other words, theresidue number or residue position of a given polymer is designated withrespect to the reference sequence rather than by the actual numericalposition of the residue within the given amino acid or polynucleotidesequence. For example, a given amino acid sequence, such as that of anengineered ketoreductase, can be aligned to a reference sequence byintroducing gaps to optimize residue matches between the two sequences.In these cases, although the gaps are present, the numbering of theresidue in the given amino acid or polynucleotide sequence is made withrespect to the reference sequence to which it has been aligned. As usedherein, a reference to a residue position, such as “Xn” as furtherdescribed below, is to be construed as referring to “a residuecorresponding to”, unless specifically denoted otherwise. Thus, forexample, “X94” refers to any amino acid at position 94 in a polypeptidesequence (e.g., SEQ ID NOS:2, 4, 10, 26, or 42).

As used herein, “stereoselectivity” refers to the preferential formationin a chemical or enzymatic reaction of one stereoisomer over anotherstereoisomer or another set of stereoisomers. Stereoselectivity can bepartial, where the formation of a stereoisomer is favored over another,or it may be complete where only one stereoisomer is formed. When thestereoisomers are enantiomers, the stereoselectivity is referred to asenantioselectivity, the fraction (typically reported as a percentage) ofone enantiomer in the sum of both enantiomers. It is commonlyalternatively reported in the art (typically as a percentage) as theenantiomeric excess (e.e.) calculated therefrom according to the formula[major enantiomer—minor enantiomer]/[major enantiomer+minor enantiomer].Where the stereoisomers are diastereoisomers, the stereoselectivity isreferred to as diastereoselectivity, the fraction (typically reported asa percentage) of one diastereomer in a mixture of two diastereomers,commonly alternatively reported as the diastereomeric excess (d.e.).Enantiomeric excess and diastereomeric excess are types of stereomericexcess. It is also to be understood that stereoselectivity is notlimited to single stereoisomers and can be described for sets ofstereoisomers.

As used herein, “highly stereoselective” refers to a chemical orenzymatic reaction that is capable of converting a substrate to itscorresponding chiral alcohol product, with at least about 75%stereomeric excess.

As used herein, “increased enzymatic activity” and “increased activity”refer to an improved property of an engineered enzyme, which can berepresented by an increase in specific activity (e.g., productproduced/time/weight protein) or an increase in percent conversion ofthe substrate to the product (e.g., percent conversion of startingamount of substrate to product in a specified time period using aspecified amount of ketoreductase) as compared to a reference enzyme.Exemplary methods to determine enzyme activity are provided in theExamples. Any property relating to enzyme activity may be affected,including the classical enzyme properties of Km, Vmax or kcat, changesof which can lead to increased enzymatic activity. The ketoreductaseactivity can be measured by any one of standard assays used formeasuring ketoreductases, such as change in substrate or productconcentration, or change in concentration of the cofactor (in absence ofa cofactor regenerating system). Comparisons of enzyme activities aremade using a defined preparation of enzyme, a defined assay under a setcondition, and one or more defined substrates, as further described indetail herein. Generally, when enzymes in cell lysates are compared, thenumbers of cells and the amount of protein assayed are determined aswell as use of identical expression systems and identical host cells tominimize variations in amount of enzyme produced by the host cells andpresent in the lysates.

As used herein, “conversion” refers to the enzymatic transformation of asubstrate to the corresponding product.

As used herein “percent conversion” refers to the percent of thesubstrate that is converted to the product within a period of time underspecified conditions. Thus, for example, the “enzymatic activity” or“activity” of a ketoreductase polypeptide can be expressed as “percentconversion” of the substrate to the product.

As used herein, “thermostable” or “thermal stable” are usedinterchangeably to refer to a polypeptide that is resistant toinactivation when exposed to a set of temperature conditions (e.g.,40-80° C.) for a period of time (e.g., 0.5-24 hrs) compared to theuntreated enzyme, thus retaining a certain level of residual activity(e.g., more than 60% to 80% for example) after exposure to elevatedtemperatures.

As used herein, “solvent stable” refers to the ability of a polypeptideto maintain similar activity (e.g., more than e.g., 60% to 80%) afterexposure to varying concentrations (e.g., 5-99%) of solvent (e.g.,isopropyl alcohol, tetrahydrofuran, 2-methyltetrahydrofuran, acetone,toluene, butylacetate, methyl tert-butylether, etc.) for a period oftime (e.g., 0.5-24 hrs) compared to the untreated enzyme.

As used herein, “amino acid difference” or “residue difference” refersto a difference in the amino acid residue at a position of a polypeptidesequence relative to the amino acid residue at a corresponding positionin a reference sequence. The positions of amino acid differencesgenerally are referred to herein as “Xn,” where n refers to thecorresponding position in the reference sequence upon which the residuedifference is based. For example, a “residue difference at position X40as compared to SEQ ID NO:2” refers to a difference of the amino acidresidue at the polypeptide position corresponding to position 40 of SEQID NO:2. Thus, if the reference polypeptide of SEQ ID NO:2 has ahistidine at position 40, then a “residue difference at position X40 ascompared to SEQ ID NO:2” refers to an amino acid substitution of anyresidue other than histidine at the position of the polypeptidecorresponding to position 40 of SEQ ID NO:2. In most instances herein,the specific amino acid residue difference at a position is indicated as“XnY” where “Xn” specified the corresponding position as describedabove, and “Y” is the single letter identifier of the amino acid foundin the engineered polypeptide (i.e., the different residue than in thereference polypeptide). In some instances, the present disclosure alsoprovides specific amino acid differences denoted by the conventionalnotation “AnB”, where A is the single letter identifier of the residuein the reference sequence, “n” is the number of the residue position inthe reference sequence, and B is the single letter identifier of theresidue substitution in the sequence of the engineered polypeptide. Insome instances, a polypeptide of the present disclosure can include oneor more amino acid residue differences relative to a reference sequence,which is indicated by a list of the specified positions where residuedifferences are present relative to the reference sequence. In someembodiments, where more than one amino acid can be used in a specificresidue position of a polypeptide, the various amino acid residues thatcan be used are separated by a “/” (e.g., X192A/G). The presentdisclosure includes engineered polypeptide sequences comprising one ormore amino acid differences that include either/or both conservative andnon-conservative amino acid substitutions. The amino acid sequences ofthe specific recombinant carbonic anhydrase polypeptides included in theSequence Listing of the present disclosure include an initiatingmethionine (M) residue (i.e., M represents residue position 1). Theskilled artisan, however, understands that this initiating methionineresidue can be removed by biological processing machinery, such as in ahost cell or in vitro translation system, to generate a mature proteinlacking the initiating methionine residue, but otherwise retaining theenzyme's properties. Consequently, the term “amino acid residuedifference relative to SEQ ID NO:2 at position Xn” as used herein mayrefer to position “Xn” or to the corresponding position (e.g., position(X−1)n) in a reference sequence that has been processed so as to lackthe starting methionine.

As used herein, the phrase “conservative amino acid substitutions”refers to the interchangeability of residues having similar side chains,and thus typically involves substitution of the amino acid in thepolypeptide with amino acids within the same or similar defined class ofamino acids. By way of example and not limitation, in some embodiments,an amino acid with an aliphatic side chain is substituted with anotheraliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine);an amino acid with a hydroxyl side chain is substituted with anotheramino acid with a hydroxyl side chain (e.g., serine and threonine); anamino acids having aromatic side chains is substituted with anotheramino acid having an aromatic side chain (e.g., phenylalanine, tyrosine,tryptophan, and histidine); an amino acid with a basic side chain issubstituted with another amino acid with a basis side chain (e.g.,lysine and arginine); an amino acid with an acidic side chain issubstituted with another amino acid with an acidic side chain (e.g.,aspartic acid or glutamic acid); and/or a hydrophobic or hydrophilicamino acid is replaced with another hydrophobic or hydrophilic aminoacid, respectively. Exemplary conservative substitutions are provided inTable 1.

TABLE 1 Exemplary Conservative Amino Acid Substitutions Residue PossibleConservative Substitutions A, L, V, I Other aliphatic (A, L, V, I) Othernon-polar (A, L, V, I, G, M) G, M Other non-polar (A, L, V, I, G, M) D,E Other acidic (D, E) K, R Other basic (K, R) N, Q, S, T Other polar H,Y, W, F Other aromatic (H, Y, W, F) C, P Non-polar

As used herein, the phrase “non-conservative substitution” refers tosubstitution of an amino acid in the polypeptide with an amino acid withsignificantly differing side chain properties. Non-conservativesubstitutions may use amino acids between, rather than within, thedefined groups and affects (a) the structure of the peptide backbone inthe area of the substitution (e.g., proline for glycine) (b) the chargeor hydrophobicity, or (c) the bulk of the side chain. By way of exampleand not limitation, an exemplary non-conservative substitution can be anacidic amino acid substituted with a basic or aliphatic amino acid; anaromatic amino acid substituted with a small amino acid; and ahydrophilic amino acid substituted with a hydrophobic amino acid.

As used herein, “deletion” refers to modification of the polypeptide byremoval of one or more amino acids from the reference polypeptide.Deletions can comprise removal of 1 or more amino acids, 2 or more aminoacids, 5 or more amino acids, 10 or more amino acids, 15 or more aminoacids, or 20 or more amino acids, up to 10% of the total number of aminoacids, or up to 20% of the total number of amino acids making up thepolypeptide while retaining enzymatic activity and/or retaining theimproved properties of an engineered enzyme. Deletions can be directedto the internal portions and/or terminal portions of the polypeptide. Invarious embodiments, the deletion can comprise a continuous segment orcan be discontinuous.

As used herein, “insertion” refers to modification of the polypeptide byaddition of one or more amino acids to the reference polypeptide. Insome embodiments, the improved engineered ketoreductase enzymes compriseinsertions of one or more amino acids to the naturally occurringketoreductase polypeptide as well as insertions of one or more aminoacids to engineered ketoreductase polypeptides. Insertions can be in theinternal portions of the polypeptide, or to the carboxy or aminoterminus. Insertions as used herein include fusion proteins as is knownin the art. The insertion can be a contiguous segment of amino acids orseparated by one or more of the amino acids in the naturally occurringpolypeptide.

The term “amino acid substitution set” or “substitution set” refers to agroup of amino acid substitutions in a polypeptide sequence, as comparedto a reference sequence. A substitution set can have 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions. Insome embodiments, a substitution set refers to the set of amino acidsubstitutions that is present in any of the variant KREDs listed in theTables provided in the Examples.

As used herein, “fragment” refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion, but where the remainingamino acid sequence is identical to the corresponding positions in thesequence. Fragments can typically have about 80%, about 90%, about 95%,about 98%, or about 99% of the full-length ketoreductase polypeptide,for example the polypeptide of SEQ ID NO:4. In some embodiments, thefragment is “biologically active” (i.e., it exhibits the same enzymaticactivity as the full-length sequence).

As used herein, “isolated polypeptide” refers to a polypeptide which issubstantially separated from other contaminants that naturally accompanyit, e.g., protein, lipids, and polynucleotides. The term embracespolypeptides which have been removed or purified from theirnaturally-occurring environment or expression system (e.g., host cell orin vitro synthesis). The improved ketoreductase enzymes may be presentwithin a cell, present in the cellular medium, or prepared in variousforms, such as lysates or isolated preparations. As such, in someembodiments, the engineered ketoreductase polypeptides of the presentdisclosure can be an isolated polypeptide.

As used herein, “substantially pure polypeptide” refers to a compositionin which the polypeptide species is the predominant species present(i.e., on a molar or weight basis it is more abundant than any otherindividual macromolecular species in the composition), and is generallya substantially purified composition when the object species comprisesat least about 50 percent of the macromolecular species present by moleor % weight. Generally, a substantially pure engineered ketoreductasepolypeptide composition will comprise about 60% or more, about 70% ormore, about 80% or more, about 90% or more, about 91% or more, about 92%or more, about 93% or more, about 94% or more, about 95% or more, about96% or more, about 97% or more, about 98% or more, or about 99% of allmacromolecular species by mole or % weight present in the composition.Solvent species, small molecules (<500 Daltons), and elemental ionspecies are not considered macromolecular species. In some embodiments,the isolated improved ketoreductase polypeptide is a substantially purepolypeptide composition.

As used herein, when used with reference to a nucleic acid orpolypeptide, the term “heterologous” refers to a sequence that is notnormally expressed and secreted by an organism (e.g., a wild-typeorganism). In some embodiments, the term encompasses a sequence thatcomprises two or more subsequences which are not found in the samerelationship to each other as normally found in nature, or isrecombinantly engineered so that its level of expression, or physicalrelationship to other nucleic acids or other molecules in a cell, orstructure, is not normally found in nature. For instance, a heterologousnucleic acid is typically recombinantly produced, having two or moresequences from unrelated genes arranged in a manner not found in nature(e.g., a nucleic acid open reading frame (ORF) of the inventionoperatively linked to a promoter sequence inserted into an expressioncassette, such as a vector). In some embodiments, “heterologouspolynucleotide” refers to any polynucleotide that is introduced into ahost cell by laboratory techniques, and includes polynucleotides thatare removed from a host cell, subjected to laboratory manipulation, andthen reintroduced into a host cell.

As used herein, “codon optimized” refers to changes in the codons of thepolynucleotide encoding a protein to those preferentially used in aparticular organism such that the encoded protein is efficientlyexpressed in the organism of interest. In some embodiments, thepolynucleotides encoding the ketoreductase enzymes may be codonoptimized for optimal production from the host organism selected forexpression.

As used herein, “control sequence” is defined herein to include allcomponents, which are necessary or advantageous for the expression of apolynucleotide and/or polypeptide of the present disclosure. Eachcontrol sequence may be native or foreign to the polynucleotide ofinterest. Such control sequences include, but are not limited to, aleader, polyadenylation sequence, propeptide sequence, promoter, signalpeptide sequence, and transcription terminator.

As used herein, “operably linked” is defined herein as a configurationin which a control sequence is appropriately placed (i.e., in afunctional relationship) at a position relative to a polynucleotide ofinterest such that the control sequence directs or regulates theexpression of the polynucleotide and/or polypeptide of interest.

As used herein, the phrases “cofactor regeneration system” and “cofactorrecycling system” refer to a set of reactants that participate in areaction that reduces the oxidized form of the cofactor (e.g., NADP+ toNADPH). Cofactors oxidized by the ketoreductase-catalyzed reduction ofthe keto substrate are regenerated in reduced form by the cofactorregeneration system. Cofactor regeneration systems comprise astoichiometric reductant that is a source of reducing hydrogenequivalents and is capable of reducing the oxidized form of thecofactor. The cofactor regeneration system may further comprise acatalyst, for example an enzyme catalyst that catalyzes the reduction ofthe oxidized form of the cofactor by the reductant. Cofactorregeneration systems to regenerate NADH or NADPH from NAD+ or NADP+,respectively, are known in the art and may be used in the methodsdescribed herein.

As used herein, “secondary alcohol dehydrogenase” is used herein torefer to an NAD+ or NADP+-dependent enzyme that catalyzes the conversionof a secondary alcohol (e.g., isopropyl alcohol) and NAD+ or NADP+ to aketone and NADH or NADPH, respectively.

As used herein, “suitable reaction conditions” refer to those conditionsin the biocatalytic reaction solution (e.g., ranges of enzyme loading,substrate loading, cofactor loading, temperature, pH, buffers,co-solvents, etc.) under which a ketoreductase polypeptide of thepresent disclosure is capable of converting a substrate compound to aproduct compound (e.g., conversion of compound (1) to compound (2a)).Exemplary “suitable reaction conditions” are provided in the presentdisclosure and illustrated by the Examples.

As used herein, “loading,” such as in “compound loading,” “enzymeloading,” or “cofactor loading” refers to the concentration or amount ofa component in a reaction mixture at the start of the reaction.

As used herein, “substrate” in the context of a biocatalyst mediatedprocess refers to the compound or molecule acted on by the biocatalyst.For example, an exemplary substrate for the ketoreductase biocatalyst inthe process disclosed herein is compound (1).

As used herein “product” in the context of a biocatalyst mediatedprocess refers to the compound or molecule resulting from the action ofthe biocatalyst. For example, an exemplary product for the ketoreductasebiocatalyst in the process disclosed herein is compound (2a).

As used herein, “equilibration” as used herein refers to the processresulting in a steady state concentration of chemical species in achemical or enzymatic reaction (e.g., interconversion of two species Aand B), including interconversion of stereoisomers, as determined by theforward rate constant and the reverse rate constant of the chemical orenzymatic reaction.

As used herein, “alkyl” refers to saturated hydrocarbon groups of from 1to 18 carbon atoms inclusively, either straight chained or branched,more preferably from 1 to 8 carbon atoms inclusively, and mostpreferably 1 to 6 carbon atoms inclusively. An alkyl with a specifiednumber of carbon atoms is denoted in parenthesis, e.g., (C₁-C₄)alkylrefers to an alkyl of 1 to 4 carbon atoms.

As used herein, “alkenyl” refers to groups of from 2 to 12 carbon atomsinclusively, either straight or branched containing at least one doublebond but optionally containing more than one double bond.

As used herein, “alkynyl” refers to groups of from 2 to 12 carbon atomsinclusively, either straight or branched containing at least one triplebond but optionally containing more than one triple bond, andadditionally optionally containing one or more double bonded moieties.

As used herein, “heteroalkyl, “heteroalkenyl,” and heteroalkynyl,” referto alkyl, alkenyl and alkynyl as defined herein in which one or more ofthe carbon atoms are each independently replaced with the same ordifferent heteroatoms or heteroatomic groups. Heteroatoms and/orheteroatomic groups which can replace the carbon atoms include, but arenot limited to, —O—, —S—, —S—O—, —NR^(α)—, —PH—, —S(O)—, —S(O)2-, —S(O)NR^(α)—, —S(O)₂NR^(α)—, and the like, including combinations thereof,where each R^(α) is independently selected from hydrogen, alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

As used herein, “alkoxy” refers to the group —OR^(β) wherein R^(β) is analkyl group is as defined above including optionally substituted alkylgroups as also defined herein.

As used herein, “aryl” refers to an unsaturated aromatic carbocyclicgroup of from 6 to 12 carbon atoms inclusively having a single ring(e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl).Exemplary aryls include phenyl, pyridyl, naphthyl and the like.

As used herein, “amino” refers to the group —NH₂. Substituted aminorefers to the group —NHR^(δ), NR^(δ)R^(δ), and NR^(δ)R^(δ)R^(δ), whereeach R^(δ) is independently selected from substituted or unsubstitutedalkyl, cycloalkyl, cycloheteroalkyl, alkoxy, aryl, heteroaryl,heteroarylalkyl, acyl, alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, andthe like. Typical amino groups include, but are limited to,dimethylamino, diethylamino, trimethylammonium, triethylammonium,methylysulfonylamino, furanyl-oxy-sulfamino, and the like.

As used herein, “oxo” refers to ═O.

As used herein, “oxy” refers to a divalent group —O—, which may havevarious substituents to form different oxy groups, including ethers andesters.

As used herein, “carboxy” refers to —COOH.

As used herein, “carbonyl” refers to —C(O)—, which may have a variety ofsubstituents to form different carbonyl groups including acids, acidhalides, aldehydes, amides, esters, and ketones.

As used herein, “alkyloxycarbonyl” refers to —C(O)OR^(ε), where R^(ε) isan alkyl group as defined herein, which can be optionally substituted.

As used herein, “aminocarbonyl” refers to —C(O)NH₂. Substitutedaminocarbonyl refers to —C(O)NR^(δ)R^(δ), where the amino groupNR^(δ)R^(δ) is as defined herein.

As used herein, “halogen” and “halo” refer to fluoro, chloro, bromo andiodo.

As used herein, “hydroxy” refers to —OH.

As used herein, “cyano” refers to —CN.

As used herein, “heteroaryl” refers to an aromatic heterocyclic group offrom 1 to 10 carbon atoms inclusively and 1 to 4 heteroatoms inclusivelyselected from oxygen, nitrogen and sulfur within the ring. Suchheteroaryl groups can have a single ring (e.g., pyridyl or furyl) ormultiple condensed rings (e.g., indolizinyl or benzothienyl).

As used herein, “heteroarylalkyl” refers to an alkyl substituted with aheteroaryl (i.e., heteroaryl-alkyl-groups), preferably having from 1 to6 carbon atoms inclusively in the alkyl moiety and from 5 to 12 ringatoms inclusively in the heteroaryl moiety. Such heteroarylalkyl groupsare exemplified by pyridylmethyl and the like.

As used herein, “heteroarylalkenyl” refers to an alkenyl substitutedwith a heteroaryl (i.e., heteroaryl-alkenyl-groups), preferably havingfrom 2 to 6 carbon atoms inclusively in the alkenyl moiety and from 5 to12 ring atoms inclusively in the heteroaryl moiety.

As used herein, “heteroarylalkynyl” refers to an alkynyl substitutedwith a heteroaryl (i.e., heteroaryl-alkynyl-groups), preferably havingfrom 2 to 6 carbon atoms inclusively in the alkynyl moiety and from 5 to12 ring atoms inclusively in the heteroaryl moiety.

As used herein, “heterocycle,” “heterocyclic,” and interchangeably“heterocycloalkyl,” refer to a saturated or unsaturated group having asingle ring or multiple condensed rings, from 2 to 10 carbon ring atomsinclusively and from 1 to 4 hetero ring atoms inclusively selected fromnitrogen, sulfur or oxygen within the ring. Such heterocyclic groups canhave a single ring (e.g., piperidinyl or tetrahydrofuryl) or multiplecondensed rings (e.g., indolinyl, dihydrobenzofuran or quinuclidinyl).Examples of heterocycles include, but are not limited to, furan,thiophene, thiazole, oxazole, pyrrole, imidazole, pyrazole, pyridine,pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole,indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, piperidine, piperazine, pyrrolidine,indoline and the like.

As used herein, “membered ring” is meant to embrace any cyclicstructure. The number preceding the term “membered” denotes the numberof skeletal atoms that constitute the ring. Thus, for example,cyclohexyl, pyridine, pyran and thiopyran are 6-membered rings andcyclopentyl, pyrrole, furan, and thiophene are 5-membered rings.

Unless otherwise specified, positions occupied by hydrogen in theforegoing groups can be further substituted with substituentsexemplified by, but not limited to, hydroxy, oxo, nitro, methoxy,ethoxy, alkoxy, substituted alkoxy, trifluoromethoxy, haloalkoxy,fluoro, chloro, bromo, iodo, halo, methyl, ethyl, propyl, butyl, alkyl,alkenyl, alkynyl, substituted alkyl, trifluoromethyl, haloalkyl,hydroxyalkyl, alkoxyalkyl, thio, alkylthio, acyl, carboxy,alkoxycarbonyl, carboxamido, substituted carboxamido, alkylsulfonyl,alkylsulfinyl, alkylsulfonylamino, sulfonamido, substituted sulfonamido,cyano, amino, substituted amino, alkylamino, dialkylamino, aminoalkyl,acylamino, amidino, amidoximo, hydroxamoyl, phenyl, aryl, substitutedaryl, aryloxy, arylalkyl, arylalkenyl, arylalkynyl, pyridyl, imidazolyl,heteroaryl, substituted heteroaryl, heteroaryloxy, heteroarylalkyl,heteroarylalkenyl, heteroarylalkynyl, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl,substituted cycloalkyl, cycloalkyloxy, pyrrolidinyl, piperidinyl,morpholino, heterocycle, (heterocycle)oxy, and (heterocycle)alkyl; andpreferred heteroatoms are oxygen, nitrogen, and sulfur. It is understoodthat where open valences exist on these substituents they can be furthersubstituted with alkyl, cycloalkyl, aryl, heteroaryl, and/or heterocyclegroups, that where these open valences exist on carbon they can befurther substituted by halogen and by oxygen-, nitrogen-, orsulfur-bonded substituents, and where multiple such open valences exist,these groups can be joined to form a ring, either by direct formation ofa bond or by formation of bonds to a new heteroatom, preferably oxygen,nitrogen, or sulfur. It is further understood that the abovesubstitutions can be made provided that replacing the hydrogen with thesubstituent does not introduce unacceptable instability to the moleculesof the present invention, and is otherwise chemically reasonable.

As used herein, “optional” and “optionally” means that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where the event or circumstance occursand instances in which it does not. One of ordinary skill in the artwould understand that with respect to any molecule described ascontaining one or more optional substituents, only sterically practicaland/or synthetically feasible compounds are meant to be included.

As used herein, “optionally substituted” refers to all subsequentmodifiers in a term or series of chemical groups. For example, in theterm “optionally substituted arylalkyl, the “alkyl” portion and the“aryl” portion of the molecule may or may not be substituted, and forthe series “optionally substituted alkyl, cycloalkyl, aryl andheteroaryl,” the alkyl, cycloalkyl, aryl, and heteroaryl groups,independently of the others, may or may not be substituted.

As used herein, “protecting group” refers to a group of atoms that mask,reduce or prevent the reactivity of the functional group when attachedto a reactive functional group in a molecule. Typically, a protectinggroup may be selectively removed as desired during the course of asynthesis. Examples of protecting groups are well-known in the art.Functional groups that can have a protecting group include, but are notlimited to, hydroxy, amino, and carboxy groups. Representative aminoprotecting groups include, but are not limited to, formyl, acetyl,trifluoroacetyl, benzyl, benzyloxycarbonyl (“CBZ”), tert-butoxycarbonyl(“Boc”), trimethylsilyl (“TMS”), 2-trimethylsilyl-ethanesulfonyl(“SES”), trityl and substituted trityl groups, allyloxycarbonyl,9-fluorenylmethyloxycarbonyl (“FMOC”), nitro-veratryloxycarbonyl(“NVOC”) and the like. Representative hydroxyl protecting groupsinclude, but are not limited to, those where the hydroxyl group iseither acylated (e.g., methyl and ethyl esters, acetate or propionategroups or glycol esters) or alkylated such as benzyl and trityl ethers,as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers(e.g., TMS or TIPPS groups) and allyl ethers. Other protecting groupscan be found in the references noted herein.

Engineered Ketoreductase Polypeptides

Ketoreductase (KRED) or carbonyl reductase biocatalysts (EC 1.1.1.184)are useful for the synthesis of alcohols from aldehydes and ketones, andoptically active secondary alcohols from the correspondingprostereoisomeric ketone substrates. KREDs may also catalyze the reversereaction, (i.e., oxidation of an alcohol substrate to the correspondingaldehydes/ketone product). The reduction of aldehydes and ketones andthe oxidation of alcohols by KREDs uses a co-factor, most commonlyreduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamideadenine dinucleotide phosphate (NADPH), and nicotinamide adeninedinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate(NADP+) for the oxidation reaction. NADH and NADPH serve as electrondonors, while NAD+ and NADP+serve as electron acceptors.

KREDs can be found in a wide range of bacteria and yeasts, as known inthe art (See e.g., Hummel and Kula Eur. J. Biochem., 184:1-13 [1989]).Numerous KRED genes and enzyme sequences have been reported, includingthose of Candida magnoliae (Genbank Acc. No. JC7338; GI:11360538);Candida parapsilosis (Genbank Acc. No. BAA24528.1; GI:2815409),Sporobolomyces salmonicolor (Genbank Acc. No. AF160799; GI:6539734),Lactobacillus kefir (Genbank Acc. No. AAP94029.1; GI: 33112056),Lactobacillus brevis (Genbank Acc. No. 1NXQ_A; GI: 30749782), andThermoanaerobium brockii (Genbank Acc. No. P14941; GI: 1771790).

The stereoselectivity of ketoreductases have been applied to thepreparation of important pharmaceutical building blocks (See e.g.,Broussy et al., Org. Lett., 11:305-308 [2009]). Specific applications ofnaturally occurring or engineered KREDs in biocatalytic processes togenerate useful chemical compounds have been demonstrated for reductionof 4-chloroacetoacetate esters (See e.g,. Zhou, J. Am. Chem. Soc.,105:5925-5926 [1983]; Santaniello, J. Chem. Res., (S)132-133 [1984];U.S. Pat. Nos. 5,559,030; 5,700,670; and 5,891,685), reduction ofdioxocarboxylic acids (See e.g., U.S. Pat. No. 6,399,339), reduction oftert-butyl (S)-chloro-5-hydroxy-3-oxohexanoate (See e.g., U.S. Pat. No.6,645,746; and WO 01/40450), reduction pyrrolotriazine-based compounds(See e.g., U.S. Appln. Publ. No. 2006/0286646); reduction of substitutedacetophenones (See e.g., U.S. Pat. Nos. 6,800,477 and 8,748,143); andreduction of ketothiolanes (WO 2005/054491).

The present invention provides engineered ketoreductases capable ofusing the substrate compound (1), tert-butyl2-(3-chloro-7-oxo-6,7-dihydro-5H-cyclopenta[c]pyridin-6-yl)acetate, as asubstrate for the synthesis of product (2), tert-butyl2-06S,7S)-3-chloro-7-hydroxy-6,7-dihydro-5H-cyclopenta[c]pyridin-6-yl)acetate,or corresponding structural analogs, as shown in the following reactionand FIG. 1.

The present invention further provides improved ketoreductase enzymesand methods for using the engineered ketoreductase enzymes to synthesizechiral compounds.

Compound (1) has one chiral center and can exist in two differentdiastereomeric forms (1a and 1b). The epimerization process happensspontaneously at room temperature and at neutral to basic conditions.Consequently, the reaction by ketoreductase can result in at least fourdifferent stereomeric products (2a-2d), as shown in FIG. 2 and below.

However, (2a) is the only desired product among the four stereoisomers.The evolution program used in the development of the present inventionwas designed to improve the selectivity of the ketoreductase for the useof substrate (1) to product (2a) with minimal amount of other sideproducts (2b-2d). In addition, the enzyme variants were assessed withregard to their activity and stability.

The ketoreductase polypeptide of SEQ ID NO:4 was selected as the initialbackbone for development of the improved enzymes provided by the presentinvention. This enzyme was chosen as the starting backbone as only twoproducts (2a and 2c) were produced when it was used in the reaction; noformation of the other two products (2b and 2d) was observed, unlike thereaction products produced using the a variant enzyme (SEQ ID NO:4). Asa result, selectivity values obtained in high throughput screening arecalculated herein either by diastereomeric excess (d.e.), according toequation (1), or by diastereomeric ratio (d.r.), which is calculatedaccording to equation (2), both of which are provided below. Due to thevarability of d.e. and d.r. measurements over time selectivityimprovements are reported as the fold improvement of d.r. over thevariant enzyme (SEQ ID NO:4)

[(2a amount)−(2c amount)]/[(2a amount)+(2c amount)]  (1)

(2a amount)/(2c amount)  (2)

Indeed, the non-naturally occurring polypeptides of the presentinvention are ketoreductases engineered to have improved properties ascompared to the naturally occurring ketoreductase of SEQ ID NO:2 and/orthe engineered ketoreductase of SEQ ID NO:4.

In some embodiments, the engineered ketoreductase polypeptides arecapable of converting the substrate compound to product with an activitythat is increased at least about 1.2 fold, 1.5 fold, 2 fold, 3 fold, 4fold, 5 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, or 100 foldrelative to the activity of the reference polypeptide of SEQ ID NO:4under suitable reaction conditions. In some embodiments, the engineeredketoreductase polypeptides are capable of converting the substratecompound to product with a percent conversion of at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, or at least about 90%, at least about 95%, at least about 98%, atleast about 99%, in a reaction time of about 48 h, about 36 h, about 24h, or even shorter length of time, under suitable reaction conditions.

In some embodiments, the engineered ketoreductases are capable ofconverting substrate compound (1) to product compound (2a) indiastereomeric excess over compound (2c). In some embodiments, theengineered ketoreductases are capable of converting compound (1) tocompound (2a) in diastereomeric excess of at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or greater over compound (2c) under suitablereaction conditions.

As will be appreciated by those of skill in the art, some of theabove-defined categories, unless otherwise specified, are not mutuallyexclusive. Thus, amino acids having side chains exhibiting two or morephysico-chemical properties can be included in multiple categories. Theappropriate classification of any amino acid or residue will be apparentto those of skill in the art, especially in light of the detaileddisclosure provided herein.

In some embodiments, the improved engineered ketoreductase enzymescomprise deletions of the naturally occurring ketoreductase polypeptidesor deletions of other engineered ketoreductase polypeptides. In someembodiments, each of the improved engineered ketoreductase enzymesdescribed herein can comprise deletions of the polypeptides describedherein. Thus, for each and every embodiment of the ketoreductasepolypeptides of the disclosure, the deletions can comprise one or moreamino acids, 2 or more amino acids, 3 or more amino acids, 4 or moreamino acids, 5 or more amino acids, 6 or more amino acids, 8 or moreamino acids, 10 or more amino acids, 15 or more amino acids, or 20 ormore amino acids, up to 10% of the total number of amino acids, up to10% of the total number of amino acids, up to 20% of the total number ofamino acids, or up to 30% of the total number of amino acids of theketoreductase polypeptides, as long as the functional activity of theketoreductase activity is maintained. In some embodiments, the deletionscan comprise, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12,1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-25, 1-30, 1-35 or about 1-40amino acid residues. In some embodiments, the number of deletions can be1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26,30, 35 or about 40 amino acids. In some embodiments, the deletions cancomprise deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 18, or 20 amino acid residues.

As described herein, the ketoreductase polypeptides of the disclosurecan be in the form of fusion polypeptides in which the ketoreductasespolypeptides are fused to other polypeptides, such as antibody tags(e.g., myc epitope) or purifications sequences (e.g., His tags). Thus,the ketoreductase polypeptides can be used with or without fusions toother polypeptides.

In some embodiments, the polypeptides described herein are notrestricted to the genetically encoded amino acids. In addition to thegenetically encoded amino acids, the polypeptides described herein maybe comprised, either in whole or in part, of naturally-occurring and/orsynthetic non-encoded amino acids. Certain commonly encounterednon-encoded amino acids of which the polypeptides described herein maybe comprised include, but are not limited to: the D-stereomers of thegenetically-encoded amino acids; 2,3-diaminopropionic acid (Dpr);α-aminoisobutyric acid (Aib); ε-aminohexanoic acid (Aha); δ-aminovalericacid (Ava); N-methylglycine or sarcosine (MeGly or Sar); ornithine(Orn); citrulline (Cit); t-butylalanine (Bua); t-butylglycine (Bug);N-methylisoleucine (MeIle); phenylglycine (Phg); cyclohexylalanine(Cha); norleucine (Nle); naphthylalanine (Nal); 2-chlorophenylalanine(Ocf); 3-chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf);2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff);4-fluorophenylalanine (Pff); 2-bromophenylalanine (Obf);3-bromophenylalanine (Mbf); 4-bromophenylalanine (Pbf);2-methylphenylalanine (Omf); 3-methylphenylalanine (Mmf);4-methylphenylalanine (Pmf); 2-nitrophenylalanine (Onf);3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf);2-cyanophenylalanine (Ocf); 3-cyanophenylalanine (Mcf);4-cyanophenylalanine (Pcf); 2-trifluoromethylphenylalanine (Otf);3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine(Ptf); 4-aminophenylalanine (Paf); 4-iodophenylalanine (Pif);4-aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef);3,4-dichlorophenylalanine (Mpcf); 2,4-difluorophenylalanine (Opff);3,4-difluorophenylalanine (Mpff); pyrid-2-ylalanine (2pAla);pyrid-3-ylalanine (3pAla); pyrid-4-ylalanine (4pAla); naphth-1-ylalanine(1nAla); naphth-2-ylalanine (2nAla); thiazolylalanine (taAla);benzothienylalanine (bAla); thienylalanine (tAla); furylalanine (fAla);homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp);pentafluorophenylalanine (5f0; styrylkalanine (sAla); authrylalanine(aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenypentanoic acid (Afp);penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic); β-2-thienylalanine (Thi); methionine sulfoxide (Mso);N(w)-nitroarginine (nArg); homolysine (hLys);phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer);phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutanic acid(hGlu); 1-aminocyclopent-(2 or 3)-ene-4 carboxylic acid; pipecolic acid(PA), azetidine-3-carboxylic acid (ACA);1-aminocyclopentane-3-carboxylic acid; allylglycine (aOly);propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal);homoleucine (hLeu), homovaline (hVal); homoisolencine (hIle);homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid(Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal);homocysteine (hCys); homoserine (hSer); hydroxyproline (Hyp) andhomoproline (hPro). Additional non-encoded amino acids of which thepolypeptides described herein may be comprised are apparent to those ofskill in the art. These amino acids may be in either the L- orD-configuration.

Those of skill in the art will recognize that amino acids or residuesbearing side chain protecting groups may also comprise the polypeptidesdescribed herein. Non-limiting examples of such protected amino acids,which in this case belong to the aromatic category, include (protectinggroups listed in parentheses), but are not limited to: Arg(tos),Cys(methylbenzyl), Cys (nitropyridinesulfenyl), Glu(δ-benzylester),Gln(xanthyl), Asn(N-δ-xanthyl), His(bom), His(benzyl), His(tos),Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr (O-benzyl) and Tyr(O-benzyl).

Non-encoding amino acids that are conformationally constrained of whichthe polypeptides described herein may be composed include, but are notlimited to, N-methyl amino acids (L-configuration); 1-aminocyclopent-(2or 3)-ene-4-carboxylic acid; pipecolic acid; azetidine-3-carboxylicacid; homoproline (hPro); and 1-aminocyclopentane-3-carboxylic acid.

As described above the various modifications introduced into thenaturally occurring polypeptide to generate an engineered ketoreductaseenzyme can be targeted to a specific property of the enzyme.

Polynucleotides Encoding Engineered Ketoreductases

In another aspect, the present disclosure provides polynucleotidesencoding the engineered ketoreductase enzymes. The polynucleotides maybe operatively linked to one or more heterologous regulatory sequencesthat control gene expression to create a recombinant polynucleotidecapable of expressing the polypeptide. Expression constructs containinga heterologous polynucleotide encoding the engineered ketoreductase canbe introduced into appropriate host cells to express the correspondingketoreductase polypeptide.

Because of the knowledge of the codons corresponding to the variousamino acids, availability of a protein sequence provides a descriptionof all the polynucleotides capable of encoding the subject. Thedegeneracy of the genetic code, where the same amino acids are encodedby alternative or synonymous codons allows an extremely large number ofnucleic acids to be made, all of which encode the improved ketoreductaseenzymes disclosed herein. Thus, having identified a particular aminoacid sequence, those skilled in the art could make any number ofdifferent nucleic acids by simply modifying the sequence of one or morecodons in a way which does not change the amino acid sequence of theprotein. In this regard, the present disclosure specificallycontemplates each and every possible variation of polynucleotides thatcould be made by selecting combinations based on the possible codonchoices, and all such variations are to be considered specificallydisclosed for any polypeptide disclosed herein, including the amino acidsequences presented in the Tables in the Examples. In variousembodiments, the codons are preferably selected to fit the host cell inwhich the protein is being produced. For example, preferred codons usedin bacteria are used to express the gene in bacteria; preferred codonsused in yeast are used for expression in yeast; and preferred codonsused in mammals are used for expression in mammalian cells. By way ofexample, the polynucleotide of SEQ ID NO:2 has been codon optimized forexpression in E. coli, but otherwise encodes the naturally occurringketoreductase of Lactobacillus kefir.

In some embodiments, the polynucleotide comprises a nucleotide sequenceencoding a ketoreductase polypeptide with an amino acid sequence thathas at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% or more sequence identity to any of thereference engineered ketoreductase polypeptides described herein, wherethe encoded ketoreductase polypeptide comprises an amino acid sequencein which the residue corresponding to X190 of SEQ ID NOS:4, 6, 25, 42 or238 is not a tyrosine. In some embodiments, the polynucleotide encodes aketoreductase polypeptide comprising an amino acid sequence in which theresidue corresponding to X190 is a non-aromatic residue. In someembodiments, the polynucleotide encodes a ketoreductase polypeptidecomprising an amino acid sequence in which the residue corresponding toX190 is alanine, glutamine, threonine, or proline, particularly proline.In some embodiments, the polynucleotide encodes an engineeredketoreductase polypeptide comprising an amino acid sequence selectedfrom SEQ ID NOS:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214,216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, and 238.

In some embodiments, the polynucleotides encoding the engineeredketoreductases are selected from SEQ ID NOS:5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55,57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91,93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121,123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149,151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177,179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205,207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233,235, and 237.

In some embodiments, the polynucleotides are capable of hybridizingunder highly stringent conditions to a polynucleotide comprising SEQ IDNO:4, wherein the polynucleotide hybridizing under highly stringentconditions has (S)-selectivity in reducing or converting the substrateof structural formula (I) to the product of structural formula (II). Insome embodiments, the polynucleotides hybridizing under highly stringentconditions are capable of reducing or converting the substrate ofstructural formula (III) to the product of structural formula (IV).

In some embodiments, the polynucleotides encode the polypeptidesdescribed herein but have about 80% or more sequence identity, about85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% or more sequence identity at the nucleotide level to a referencepolynucleotide encoding the engineered ketoreductase. In someembodiments, the reference polynucleotide is selected from apolynucleotide sequence corresponding to SEQ ID NO:4.

In some embodiments, the engineered ketoreductase sequences comprisesequences that comprise positions identified to be beneficial, asdescribed in the Examples. In some embodiments, the sequences are SEQ IDNO:239 or SEQ ID NO:240. In some further embodiments, the sequencescomprise variant sequences derived from SEQ ID NO:239 or SEQ ID NO:240(i.e., SEQ ID NO:239 or SEQ ID NO:240 is the starting sequence fromwhich the variant sequences are developed).

An isolated polynucleotide encoding an improved ketoreductasepolypeptide may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the isolatedpolynucleotide prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotides and nucleic acid sequences utilizingrecombinant DNA methods are well known in the art.

For bacterial host cells, suitable promoters for directing transcriptionof the nucleic acid constructs of the present disclosure, include thepromoters obtained from the E. coli lac operon, Streptomyces coelicoloragarase gene (dagA), Bacillus subtilis levansucrase gene (sacB),Bacillus licheniformis alpha-amylase gene (amyL), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillusamyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformispenicillinase gene (penP), Bacillus subtilis xy1A and xy1B genes, andprokaryotic beta-lactamase gene (See e.g., Villa-Kamaroff et al., Proc.Natl. Acad. Sci. USA 75: 3727-3731 [1978]), as well as the tac promoter(See e.g., DeBoer et al., Proc. Natl Acad. Sci. USA 80: 21-25 [1983]).Additional suitable promoters are known to those in the art.

For filamentous fungal host cells, suitable promoters for directing thetranscription of the nucleic acid constructs of the present disclosureinclude promoters obtained from the genes for Aspergillus oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, andFusarium oxysporum trypsin-like protease (WO 96/00787), as well as theNA2-tpi promoter (a hybrid of the promoters from the genes forAspergillus niger neutral alpha-amylase and Aspergillus oryzae triosephosphate isomerase), and mutant, truncated, and hybrid promotersthereof.

In a yeast host, useful promoters include, but are not limited to thosefrom the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiaealcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase(ADH2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase, aswell as other useful promoters for yeast host cells (See e.g., Romanoset al., Yeast 8:423-488 [1992]).

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator that is functional in the host cell of choice may be used inthe present invention.

For example, exemplary transcription terminators for filamentous fungalhost cells can be obtained from the genes for Aspergillus oryzae TAKAamylase, Aspergillus niger glucoamylase, Aspergillus nidulansanthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusariumoxysporum trypsin-like protease.

Exemplary terminators for yeast host cells can be obtained from thegenes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase, as well as other usefulterminators for yeast host cells known in the art (See e.g,. Romanos etal., supra).

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′ terminus ofthe nucleic acid sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used. Exemplaryleaders for filamentous fungal host cells are obtained from the genesfor Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leaders for yeast host cells are obtainedfrom the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomycescerevisiae alpha-factor, and Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleic acid sequence andwhich, when transcribed, is recognized by the host cell as a signal toadd polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention. Exemplary polyadenylation sequences forfilamentous fungal host cells can be from the genes for Aspergillusoryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillusnidulans anthranilate synthase, Fusarium oxysporum trypsin-likeprotease, and Aspergillus niger alpha-glucosidase, as well as additionaluseful polyadenylation sequences for yeast host cells known in the art(See e.g., Guo et al., Mol. Cell. Biol., 15:5983-5990 [1995]).

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion that encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region thatis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region.

Alternatively, the foreign signal peptide coding region may simplyreplace the natural signal peptide coding region in order to enhancesecretion of the polypeptide. However, any signal peptide coding regionwhich directs the expressed polypeptide into the secretory pathway of ahost cell of choice may be used in the present invention.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NC1B11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA, as well as additional signalpeptides known in the art (See e.g., Simonen et al., Microbiol. Rev.,57: 109-137 [1993]).

Effective signal peptide coding regions for filamentous fungal hostcells include, but are not limited to the signal peptide coding regionsobtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillusniger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor mieheiaspartic proteinase, Humicola insolens cellulase, and Humicolalanuginosa lipase. Useful signal peptides for yeast host cells can befrom the genes for Saccharomyces cerevisiae alpha-factor andSaccharomyces cerevisiae invertase, as well as additional useful signalpeptide coding regions (See e.g., Romanos et al., 1992, supra).

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalactase (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences, which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. In prokaryotic host cells, suitable regulatory sequencesinclude the lac, tac, and trp operator systems. In yeast host cells,suitable regulatory systems include, as examples, the ADH2 system orGAL1 system. In filamentous fungi, suitable regulatory sequences includethe TAKA alpha-amylase promoter, Aspergillus niger glucoamylasepromoter, and Aspergillus oryzae glucoamylase promoter.

Other examples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene, which is amplified in the presence of methotrexate, andthe metallothionein genes, which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the KRED polypeptide ofthe present invention would be operably linked with the regulatorysequence.

Thus, in some embodiments, the present disclosure is also directed to arecombinant expression vector comprising a polynucleotide encoding anengineered ketoreductase polypeptide or a variant thereof, and one ormore expression regulating regions such as a promoter and a terminator,a replication origin, etc., depending on the type of hosts into whichthey are to be introduced. The various nucleic acid and controlsequences described above may be joined together to produce arecombinant expression vector which may include one or more convenientrestriction sites to allow for insertion or substitution of the nucleicacid sequence encoding the polypeptide at such sites. Alternatively, thenucleic acid sequence of the present disclosure may be expressed byinserting the nucleic acid sequence or a nucleic acid constructcomprising the sequence into an appropriate vector for expression. Increating the expression vector, the coding sequence is located in thevector so that the coding sequence is operably linked with theappropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus), which can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the polynucleotidesequence. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

The expression vector may be an autonomously replicating vector (i.e., avector that exists as an extrachromosomal entity), the replication ofwhich is independent of chromosomal replication, (e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificialchromosome). The vector may contain any means for assuringself-replication. Alternatively, the vector may be one which, whenintroduced into the host cell, is integrated into the genome andreplicated together with the chromosome(s) into which it has beenintegrated. Furthermore, a single vector or plasmid or two or morevectors or plasmids which together contain the total DNA to beintroduced into the genome of the host cell, or a transposon may beused.

The expression vector of the present invention preferably contains oneor more selectable markers, which permit easy selection of transformedcells. A selectable marker can be a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like. Examples of bacterial selectable markersare the dal genes from Bacillus subtilis or Bacillus licheniformis, ormarkers, which confer antibiotic resistance such as ampicillin,kanamycin, chloramphenicol, or tetracycline resistance. Suitable markersfor yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.

Selectable markers for use in a filamentous fungal host cell include,but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricin acetyltransferase), hph(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Embodiments for use in an Aspergillus cell include the amdS and pyrGgenes of Aspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The expression vectors of the present invention can contain anelement(s) that permits integration of the vector into the host cell'sgenome or autonomous replication of the vector in the cell independentof the genome. For integration into the host cell genome, the vector mayrely on the nucleic acid sequence encoding the polypeptide or any otherelement of the vector for integration of the vector into the genome byhomologous or nonhomologous recombination.

Alternatively, the expression vector may contain additional nucleic acidsequences for directing integration by homologous recombination into thegenome of the host cell. The additional nucleic acid sequences enablethe vector to be integrated into the host cell genome at a preciselocation(s) in the chromosome(s). To increase the likelihood ofintegration at a precise location, the integrational elements shouldpreferably contain a sufficient number of nucleic acids, such as 100 to10,000 base pairs, preferably 400 to 10,000 base pairs, and mostpreferably 800 to 10,000 base pairs, which are highly homologous withthe corresponding target sequence to enhance the probability ofhomologous recombination. The integrational elements may be any sequencethat is homologous with the target sequence in the genome of the hostcell. Furthermore, the integrational elements may be non-encoding orencoding nucleic acid sequences. On the other hand, the vector may beintegrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are P15Aori or the origins of replication of plasmids pBR322, pUC19, pACYC177(which plasmid has the P15A ori), or pACYC184 permitting replication inE. coli, and pUB110, pE194, pTA1060, or pAM131 permitting replication inBacillus. Examples of origins of replication for use in a yeast hostcell are the 2 micron origin of replication, ARS1, ARS4, the combinationof ARS1 and CEN3, and the combination of ARS4 and CEN6. The origin ofreplication may be one having a mutation which makes it's functioningtemperature-sensitive in the host cell (See e.g., Ehrlich, Proc. Natl.Acad. Sci. USA 75:1433 [1978]).

More than one copy of a nucleic acid sequence of the present inventionmay be inserted into the host cell to increase production of the geneproduct. An increase in the copy number of the nucleic acid sequence canbe obtained by integrating at least one additional copy of the sequenceinto the host cell genome or by including an amplifiable selectablemarker gene with the nucleic acid sequence where cells containingamplified copies of the selectable marker gene, and thereby additionalcopies of the nucleic acid sequence, can be selected for by cultivatingthe cells in the presence of the appropriate selectable agent.

Many of the expression vectors for use in the present disclosure arecommercially available. Suitable commercial expression vectors include,but are not limited to p3×FLAG™™ expression vectors (Sigma-Aldrich),which include a CMV promoter and hGH polyadenylation site for expressionin mammalian host cells and a pBR322 origin of replication andampicillin resistance markers for amplification in E. coli. Othercommercially available suitable expression vectors include but are notlimited to the pBluescriptII SK(−) and pBK-CMV vectors (Stratagene), andplasmids derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4(Invitrogen) or pPoly (See, Lathe et al., Gene 57:193-201 [1987]).

Host Cells for Expression of Ketoreductase Polypeptides

In another aspect, the present disclosure provides a host cellcomprising a polynucleotide encoding an improved ketoreductasepolypeptide of the present disclosure, the polynucleotide beingoperatively linked to one or more control sequences for expression ofthe ketoreductase enzyme in the host cell. Host cells for use inexpressing the KRED polypeptides encoded by the expression vectors ofthe present invention are well known in the art and include but are notlimited to, bacterial cells, such as E. coli, Lactobacillus kefir,Lactobacillus brevis, Lactobacillus minor, Streptomyces and Salmonellatyphimurium cells; fungal cells, such as yeast cells (e.g.,Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No.201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells;animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; andplant cells. Appropriate culture media and growth conditions for theabove-described host cells are well known in the art.

Polynucleotides for expression of the ketoreductase may be introducedinto cells by various methods known in the art. Techniques include amongothers, electroporation, biolistic particle bombardment, liposomemediated transfection, calcium chloride transfection, and protoplastfusion. Various methods for introducing polynucleotides into cells willbe apparent to the skilled artisan.

Escherichia coli W3110 is a host strain that finds use in the presentinvention, although it is not intended that the present invention belimited to this specific host strain. The expression vector was createdby operatively linking a polynucleotide encoding an improvedketoreductase into the plasmid pCK110900 operatively linked to the lacpromoter under control of the lacI repressor. The expression vector alsocontained the P15a origin of replication and the chloramphenicolresistance gene. Cells containing the subject polynucleotide inEscherichia coli W3110 can be isolated by subjecting the cells tochloramphenicol selection.

Methods of Generating Engineered Ketoreductase Polypeptides.

In some embodiments, to make the improved KRED polynucleotides andpolypeptides of the present disclosure, the naturally-occurringketoreductase enzyme that catalyzes the reduction reaction is obtained(or derived) from Lactobacillus kefir or Lactobacillus brevis orLactobacillus minor. In some embodiments, the parent polynucleotidesequence is codon optimized to enhance expression of the ketoreductasein a specified host cell. As an illustration, the parentalpolynucleotide sequence encoding the wild-type KRED polypeptide ofLactobacillus kefir was constructed from oligonucleotides prepared basedupon the known polypeptide sequence of Lactobacillus kefir KRED sequenceavailable in Genbank database (Genbank accession no. AAP94029GI:33112056). The parental polynucleotide sequence, designated as SEQ IDNO:3, was codon optimized for expression in E. coli and thecodon-optimized polynucleotide cloned into an expression vector, placingthe expression of the ketoreductase gene under the control of the lacpromoter and lad repressor gene. Clones expressing the activeketoreductase in E. coli were identified and the genes sequenced toconfirm their identity. The sequence designated (SEQ ID NO:3) was theparent sequence utilized as the starting point for most experiments andlibrary construction of engineered ketoreductases evolved from theLactobacillus kefir ketoreductase.

The engineered ketoreductases can be obtained by subjecting thepolynucleotide encoding the naturally occurring ketoreductase tomutagenesis and/or directed evolution methods, as discussed above.Mutagenesis may be performed in accordance with any of the techniquesknown in the art, including random and site-specific mutagenesis.Directed evolution can be performed with any of the techniques known inthe art to screen for improved promoter variants including shuffling.Mutagenesis and directed evolution methods are well known in the art(See e.g., U.S. Pat. Nos. 5,605,793, 5,830,721, 6,132,970, 6,420,175,6,277,638, 6,365,408, 6,602,986, 7,288,375, 6,287,861, 6,297,053,6,576,467, 6,444,468, 5,811238, 6,117,679, 6,165,793, 6,180,406,6,291,242, 6,995,017, 6,395,547, 6,506,602, 6,519,065, 6,506,603,6,413,774, 6,573,098, 6,323,030, 6,344,356, 6,372,497, 7,868,138,5,834,252, 5,928,905, 6,489,146, 6,096,548, 6,387,702, 6,391,552,6,358,742, 6,482,647, 6,335,160, 6,653,072, 6,355,484, 6,03,344,6,319,713, 6,613,514, 6,455,253, 6,579,678, 6,586,182, 6,406,855,6,946,296, 7,534,564, 7,776,598, 5,837,458, 6,391,640, 6,309,883,7,105,297, 7,795,030, 6,326,204, 6,251,674, 6,716,631, 6,528,311,6,287,862, 6,335,198, 6,352,859, 6,379,964, 7,148,054, 7,629,170,7,620,500, 6,365,377, 6,358,740, 6,406,910, 6,413,745, 6,436,675,6,961,664, 7,430,477, 7,873,499, 7,702,464, 7,783,428, 7,747,391,7,747,393, 7,751,986, 6,376,246, 6,426,224, 6,423,542, 6,479,652,6,319,714, 6,521,453, 6,368,861, 7,421,347, 7,058,515, 7,024,312,7,620,502, 7,853,410, 7,957,912, 7,904,249, and all related non-UScounterparts; Ling et al., Anal. Biochem., 254(2):157-78 [1997]; Dale etal., Meth. Mol. Biol., 57:369-74 [1996]; Smith, Ann. Rev. Genet.,19:423-462 [1985]; Botstein et al., Science, 229:1193-1201 [1985];Carter, Biochem. J., 237:1-7 [1986]; Kramer et al., Cell, 38:879-887[1984]; Wells et al., Gene, 34:315-323 [1985]; Minshull et al., Curr.Op. Chem. Biol., 3:284-290 [1999]; Christians et al., Nat. Biotechnol.,17:259-264 [1999]; Crameri et al., Nature, 391:288-291 [1998]; Crameri,et al., Nat. Biotechnol., 15:436-438 [1997]; Zhang et al., Proc. Nat.Acad. Sci. U.S.A., 94:4504-4509 [1997]; Crameri et al., Nat.Biotechnol., 14:315-319 [1996]; Stemmer, Nature, 370:389-391 [1994];Stemmer, Proc. Nat. Acad. Sci. USA, 91:10747-10751 [1994]; WO 95/22625;WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767; and WO2009/152336, all of which are incorporated herein by reference).

The clones obtained following mutagenesis treatment are screened forengineered ketoreductases having a desired improved enzyme property.Measuring enzyme activity from the expression libraries can be performedusing the standard biochemistry technique of monitoring the rate ofdecrease (via a decrease in absorbance or fluorescence) of NADH or NADPHconcentration, as it is converted into NAD⁺ or NADP⁺. In this reaction,the NADH or NADPH is consumed (oxidized) by the ketoreductase as theketoreductase reduces a ketone substrate to the corresponding hydroxylgroup. The rate of decrease of NADH or NADPH concentration, as measuredby the decrease in absorbance or fluorescence, per unit time indicatesthe relative (enzymatic) activity of the KRED polypeptide in a fixedamount of the lysate (or a lyophilized powder made therefrom). Thestereochemistry of the products can be ascertained by various knowntechniques, and as provided in the Examples. Where the improved enzymeproperty desired is thermal stability, enzyme activity may be measuredafter subjecting the enzyme preparations to a defined temperature andmeasuring the amount of enzyme activity remaining after heat treatments.Clones containing a polynucleotide encoding a ketoreductase are thenisolated, sequenced to identify the nucleotide sequence changes (ifany), and used to express the enzyme in a host cell.

Where the sequence of the engineered polypeptide is known, thepolynucleotides encoding the enzyme can be prepared by standardsolid-phase methods, according to known synthetic methods. In someembodiments, fragments of up to about 100 bases can be individuallysynthesized, then joined (e.g., by enzymatic or chemical litigationmethods, or polymerase mediated methods) to form any desired continuoussequence. For example, polynucleotides and oligonucleotides of theinvention can be prepared by chemical synthesis (e.g., using theclassical phosphoramidite method described by Beaucage et al., Tet.Lett., 22:1859-69 [1981], or the method described by Matthes et al.,EMBO J., 3:801-05 [1984], as it is typically practiced in automatedsynthetic methods). According to the phosphoramidite method,oligonucleotides are synthesized (e.g., in an automatic DNAsynthesizer), purified, annealed, ligated and cloned in appropriatevectors. In addition, essentially any nucleic acid can be obtained fromany of a variety of commercial sources (e.g., The Midland CertifiedReagent Company, Midland, Tex., The Great American Gene Company, Ramona,Calif., ExpressGen Inc. Chicago, Ill., Operon Technologies Inc.,Alameda, Calif., and many others).

Engineered ketoreductase enzymes expressed in a host cell can berecovered from the cells and or the culture medium using any one or moreof the well known techniques for protein purification, including, amongothers, lysozyme treatment, sonication, filtration, salting-out,ultra-centrifugation, and chromatography. Suitable solutions for lysingand the high efficiency extraction of proteins from bacteria, such as E.coli, are commercially available under the trade name CelLytic B™(Sigma-Aldrich).

Chromatographic techniques for isolation of the ketoreductasepolypeptide include, among others, reverse phase chromatography highperformance liquid chromatography, ion exchange chromatography, gelelectrophoresis, and affinity chromatography. Conditions for purifying aparticular enzyme will depend, in part, on factors such as net charge,hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc.,and will be apparent to those having skill in the art.

In some embodiments, affinity techniques may be used to isolate theimproved ketoreductase enzymes. For affinity chromatographypurification, any antibody which specifically binds the ketoreductasepolypeptide may be used. For the production of antibodies, various hostanimals, including but not limited to rabbits, mice, rats, etc., may beimmunized by injection with the ketoreductase. The ketoreductasepolypeptide may be attached to a suitable carrier, such as BSA, by meansof a side chain functional group or linkers attached to a side chainfunctional group. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (Bacillus Calmette Guerin) and Corynebacterium parvum.

The ketoreductases may be prepared and used in the form of cellsexpressing the enzymes, as crude extracts, or as isolated or purifiedpreparations. The ketoreductases may be prepared as lyophilizates, inpowder form (e.g., acetone powders), or prepared as enzyme solutions. Insome embodiments, the ketoreductases can be in the form of substantiallypure preparations.

In some embodiments, the ketoreductase polypeptides can be attached to asolid substrate. The substrate can be a solid phase, surface, and/ormembrane. A solid support can be composed of organic polymers such aspolystyrene, polyethylene, polypropylene, polyfluoroethylene,polyethyleneoxy, and polyacrylamide, as well as co-polymers and graftsthereof. A solid support can also be inorganic, such as glass, silica,controlled pore glass (CPG), reverse phase silica or metal, such as goldor platinum. The configuration of the substrate can be in the form ofbeads, spheres, particles, granules, a gel, a membrane or a surface.Surfaces can be planar, substantially planar, or non-planar. Solidsupports can be porous or non-porous, and can have swelling ornon-swelling characteristics. A solid support can be configured in theform of a well, depression, or other container, vessel, feature, orlocation. A plurality of supports can be configured on an array atvarious locations, addressable for robotic delivery of reagents, or bydetection methods and/or instruments.

Methods of Using the Engineered Ketoreductase Enzymes and CompoundsPrepared Therewith

The ketoreductase enzymes described herein are capable of catalyzing theasymmetric reduction of the keto group in7-oxo-5,6-dihydro-5H-cyclopenta(hetero)arene substrates, optionallysubstituted at one or more of the aryl positions and the 6-position andwith nitrogen substitution in the aryl ring, to the correspondingsubstituted chiral alcohol.

In some embodiments, the ketoreductases are capable of reducing orconverting the substrate compound of structural formula (I), tert-butyl2-(3-chloro-7-oxo-5,6-dihydro-5H-cyclopenta[c]pyridin-6-yl)acetate:

to the corresponding chiral alcohol product (tert-butyl2-[(6S,7S)-3-chloro-7-hydroxy-6,7-dihydro-5H-cyclopenta[c]pyridin-6-yl]acetate)of structural formula (II):

In some embodiments, the ketoreductases described herein are capable ofreducing or converting 7-oxo-5,6-dihydro-5H-cyclopenta(hetero)arenecompounds of structural formula (III):

wherein a, b, c, and d are independently selected from carbon ornitrogen. Y is selected independently from H, halogen, NH₂, OH, alkyl,alkenyl, alkynyl, aryl, heteroaryl, alkoxy, when the associated a, b, c,or d atom is carbon. Z is independently selected from H,(CH₂)_(n)alkoxycarbonyl where n=0-6, alkyl, alkenyl, alkynyl, alkoxy,aryl, heteroaryl, to the corresponding chiral alcohol product ofstructural formula (IV):

The capability of the ketoreductases described herein to catalyze thereduction reaction of a specific further substituted7-oxo-5,6-dihydro-5H-cyclopenta(hetero)arenes, can be determined byroutine experimentation, for example by methods such as described in theExamples. However, it is not intended that the present invention belimited to these specific methods, as other methods known to thoseskilled in the art may find use.

In some embodiments, the ketoreductases described herein can be used ina method for reducing the 7-oxo-5,6-dihydro-5H-cyclopenta(hetero)arenesubstrate of formula (III) to corresponding7-hydroxy-6,7-dihydro-5H-cyclopenta(hetero)arene compound of formula(IV), where the method comprises contacting or incubating the compoundof formula (III) with a ketoreductase polypeptide described herein underreaction conditions suitable for reducing or converting the compound offormula (III) to the correspondingsubstituted7-hydroxy-6,7-dihydro-5H-cyclopenta(hetero)arene compound offormula (IV). In some embodiments of this method, the substrate isreduced to the product in greater than about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or greater diastereomeric excess.

In some embodiments, the ketoreductases described herein can be used ina method for reducing a tert-butyl2-(3-chloro-7-oxo-5,6-dihydro-5H-cyclopenta[c]pyridin-6-yl)acetatesubstrate of formula (I) to its corresponding (S)-alcohol product,(tert-butyl2-[(6S,7S)-3-chloro-7-hydroxy-6,7-dihydro-5H-cyclopenta[c]pyridin-6-yl]acetate),of formula (II), where the method comprises contacting or incubatingsubstrate tert-butyl2-(3-chloro-7-oxo-5,6-dihydro-5H-cyclopenta[c]pyridin-6-yl)acetate witha ketoreductase polypeptide described herein under reaction conditionssuitable for reducing or converting tert-butyl2-(3-chloro-7-oxo-5,6-dihydro-5H-cyclopenta[c]pyridin-6-yl)acetate to(tert-butyl2-[(6S,7S)-3-chloro-7-hydroxy-6,7-dihydro-5H-cyclopenta[c]pyridin-6-yl]acetate).In some embodiments, the substrate is reduced to the product withgreater than about 2.5 fold improved diastereomeric ratio over SEQ IDNO:4, wherein the ketoreductase polypeptide comprises an amino acidsequence based on the sequence formula of SEQ ID NOS:42, 208, 210, 212,214, 216, 218, 220, 222, 224, 226, 228, and 238.

In some embodiments, an engineered (S)-selective ketoreductase enzymederived from a wild-type Lactobacillus species ketoreductase enzyme canbe used in a method to reduce acetophenone to (S)-1-phenethanol ingreater than about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 99.9% or more stereomeric excess.

In some embodiments, the substrate is reduced to the product in greaterthan about 2.5 fold increased diastereomeric ratio over SEQ ID NO:4,wherein the ketoreductase polypeptide used in the method comprises anamino acid sequence selected from SEQ ID NO:42, 208, 210, 212, 214, 216,218, 220, 222, 224, 226, 228, and 238. In some embodiments of thismethod, at least about 95% of the substrate is reduced to the product ingreater than about 90% diastereomeric excess in less than 24 hours whenthe method is carried out with the ketoreductase polypeptide at anamount of less than about 2% by weight with respect to the amount of thetert-butyl2-(3-chloro-7-oxo-5,6-dihydro-5H-cyclopenta[c]pyridin-6-yl)acetatesubstrate.

As is known by those of skill in the art, ketoreductase-catalyzedreduction reactions typically require a cofactor. Reduction reactionscatalyzed by the engineered ketoreductase enzymes described herein alsotypically require a cofactor, although many embodiments of theengineered ketoreductases require far less cofactor than reactionscatalyzed with wild-type ketoreductase enzymes. As used herein, the term“cofactor” refers to a non-protein compound that operates in combinationwith a ketoreductase enzyme. Cofactors suitable for use with theengineered ketoreductase enzymes described herein include, but are notlimited to, NADP⁺ (nicotinamide adenine dinucleotide phosphate), NADPH(the reduced form of NADP⁺), NAD⁺ (nicotinamide adenine dinucleotide)and NADH (the reduced form of NAD⁺). Generally, the reduced form of thecofactor is added to the reaction mixture. The reduced NAD(P)H form canbe optionally regenerated from the oxidized NAD(P)⁺ form using acofactor regeneration system.

The term “cofactor regeneration system” refers to a set of reactantsthat participate in a reaction that reduces the oxidized form of thecofactor (e.g., NADP⁺ to NADPH). Cofactors oxidized by theketoreductase-catalyzed reduction of the keto substrate are regeneratedin reduced form by the cofactor regeneration system. Cofactorregeneration systems comprise a stoichiometric reductant that is asource of reducing hydrogen equivalents and is capable of reducing theoxidized form of the cofactor. The cofactor regeneration system mayfurther comprise a catalyst, for example an enzyme catalyst, thatcatalyzes the reduction of the oxidized form of the cofactor by thereductant. Cofactor regeneration systems to regenerate NADH or NADPHfrom NAD⁺ or NADP⁺, respectively, are known in the art and may be usedin the methods described herein.

Suitable exemplary cofactor regeneration systems that may be employedinclude, but are not limited to, glucose and glucose dehydrogenase,formate and formate dehydrogenase, glucose-6-phosphate andglucose-6-phosphate dehydrogenase, a secondary (e.g., isopropanol)alcohol and secondary alcohol dehydrogenase, phosphite and phosphitedehydrogenase, molecular hydrogen and hydrogenase, and the like. Thesesystems may be used in combination with either NADP⁺/NADPH or NAD⁺/NADHas the cofactor. Electrochemical regeneration using hydrogenase may alsobe used as a cofactor regeneration system. See, e.g., U.S. Pat. Nos.5,538,867 and 6,495,023, both of which are incorporated herein byreference. Chemical cofactor regeneration systems comprising a metalcatalyst and a reducing agent (for example, molecular hydrogen orformate) are also suitable. See, e.g., PCT publication WO 2000/053731,which is incorporated herein by reference.

The terms “glucose dehydrogenase” and “GDH” are used interchangeablyherein to refer to an NAD⁺ or NADP⁺-dependent enzyme that catalyzes theconversion of D-glucose and NAD⁺ or NADP⁺ to gluconic acid and NADH orNADPH, respectively. Equation (3), below, describes the glucosedehydrogenase-catalyzed reduction of NAD⁺ or NADP⁺ by glucose.

Glucose dehydrogenases that are suitable for use in the practice of themethods described herein include both naturally occurring glucosedehydrogenases, as well as non-naturally occurring glucosedehydrogenases. Naturally occurring glucose dehydrogenase encoding geneshave been reported in the literature. For example, the Bacillus subtilis61297 GDH gene was expressed in E. coli and was reported to exhibit thesame physicochemical properties as the enzyme produced in its nativehost (Vasantha et al., 1983, Proc. Natl. Acad. Sci. USA 80:785). Thegene sequence of the B. subtilis GDH gene, which corresponds to GenbankAcc. No. M12276, was reported by Lampel et al., 1986, J. Bacteriol.166:238-243, and in corrected form by Yamane et al., 1996, Microbiology142:3047-3056 as Genbank Acc. No. D50453. Naturally occurring GDH genesalso include those that encode the GDH from B. cereus ATCC 14579(Nature, 2003, 423:87-91; Genbank Acc. No. AE017013) and B. megaterium(Eur. J. Biochem., 1988, 174:485-490, Genbank Acc. No. X12370; J.Ferment. Bioeng., 1990, 70:363-369, Genbank Acc. No. GI216270). Glucosedehydrogenases from Bacillus sp. are provided in PCT publication WO2005/018579 as SEQ ID NOS: 10 and 12 (encoded by polynucleotidesequences corresponding to SEQ ID NOS: 9 and 11, respectively, of thePCT publication), the disclosure of which is incorporated herein byreference.

Non-naturally occurring glucose dehydrogenases may be generated usingknown methods, such as, for example, mutagenesis, directed evolution,and the like. GDH enzymes having suitable activity, whether naturallyoccurring or non-naturally occurring, may be readily identified usingthe assay described in Example 4 of PCT publication WO 2005/018579, thedisclosure of which is incorporated herein by reference. Exemplarynon-naturally occurring glucose dehydrogenases are provided in PCTpublication WO 2005/018579 as SEQ ID NOS: 62, 64, 66, 68, 122, 124, and126. The polynucleotide sequences that encode them are provided in PCTpublication WO 2005/018579 as SEQ ID NOS: 61, 63, 65, 67, 121, 123, and125, respectively. All of these sequences are incorporated herein byreference. Additional non-naturally occurring glucose dehydrogenasesthat are suitable for use in the ketoreductase-catalyzed reductionreactions disclosed herein are provided in U.S. application publicationNos. 2005/0095619 and 2005/0153417, the disclosures of which areincorporated herein by reference.

Glucose dehydrogenases employed in the ketoreductase-catalyzed reductionreactions described herein may exhibit an activity of at least about 10μmol/min/mg and sometimes at least about 10² μmol/min/mg or about 10³μmol/min/mg, up to about 10⁴ μmol/min/mg or higher in the assaydescribed in Example 4 of PCT publication WO 2005/018579.

The ketoreductase-catalyzed reduction reactions described herein aregenerally carried out in a solvent. Suitable solvents include water,organic solvents (e.g., ethyl acetate, butyl acetate, 1-octanol,heptane, octane, methyl t-butyl ether (MTBE), toluene, and the like),ionic liquids (e.g., 1-ethyl 4-methylimidazolium tetrafluoroborate,1-butyl-3-methylimidazolium tetrafluoroborate,1-butyl-3-methylimidazolium hexafluorophosphate, and the like). In someembodiments, aqueous solvents, including water and aqueous co-solventsystems, are used.

Exemplary aqueous co-solvent systems have water and one or more organicsolvent. In general, an organic solvent component of an aqueousco-solvent system is selected such that it does not completelyinactivate the ketoreductase enzyme. Appropriate co-solvent systems canbe readily identified by measuring the enzymatic activity of thespecified engineered ketoreductase enzyme with a defined substrate ofinterest in the candidate solvent system, utilizing an enzyme activityassay, such as those described herein.

The organic solvent component of an aqueous co-solvent system may bemiscible with the aqueous component, providing a single liquid phase, ormay be partly miscible or immiscible with the aqueous component,providing two liquid phases. Generally, when an aqueous co-solventsystem is employed, it is selected to be biphasic, with water dispersedin an organic solvent, or vice-versa. Generally, when an aqueousco-solvent system is utilized, it is desirable to select an organicsolvent that can be readily separated from the aqueous phase. Ingeneral, the ratio of water to organic solvent in the co-solvent systemis typically in the range of from about 90:10 to about 10:90 (v/v)organic solvent to water, and between 80:20 and 20:80 (v/v) organicsolvent to water. The co-solvent system may be pre-formed prior toaddition to the reaction mixture, or it may be formed in situ in thereaction vessel.

The aqueous solvent (water or aqueous co-solvent system) may bepH-buffered or unbuffered. Generally, the reduction can be carried outat a pH of about 10 or below, usually in the range of from about 5 toabout 10. In some embodiments, the reduction is carried out at a pH ofabout 9 or below, usually in the range of from about 5 to about 9. Insome embodiments, the reduction is carried out at a pH of about 8 orbelow, often in the range of from about 5 to about 8, and usually in therange of from about 6 to about 8. The reduction may also be carried outat a pH of about 7.8 or below, or 7.5 or below. Alternatively, thereduction may be carried out a neutral pH, i.e., about 7.

During the course of the reduction reactions, the pH of the reactionmixture may change. The pH of the reaction mixture may be maintained ata desired pH or within a desired pH range by the addition of an acid ora base during the course of the reaction. Alternatively, the pH may becontrolled by using an aqueous solvent that comprises a buffer. Suitablebuffers to maintain desired pH ranges are known in the art and include,for example, phosphate buffer, triethanolamine buffer, and the like.Combinations of buffering and acid or base addition may also be used.

When the glucose/glucose dehydrogenase cofactor regeneration system isemployed, the co-production of gluconic acid (pKa=3.6), as representedin equation (3) above causes the pH of the reaction mixture to drop ifthe resulting aqueous gluconic acid is not otherwise neutralized.

The pH of the reaction mixture may be maintained at the desired level bystandard buffering techniques, wherein the buffer neutralizes thegluconic acid up to the buffering capacity provided, or by the additionof a base concurrent with the course of the conversion. Combinations ofbuffering and base addition may also be used. Suitable buffers tomaintain desired pH ranges are described above. Suitable bases forneutralization of gluconic acid are organic bases, for example amines,alkoxides and the like, and inorganic bases, for example, hydroxidesalts (e.g., NaOH), carbonate salts (e.g., NaHCO₃), bicarbonate salts(e.g., K₂CO₃), basic phosphate salts (e.g., K₂HPO₄, Na₃PO₄), and thelike. The addition of a base concurrent with the course of theconversion may be done manually while monitoring the reaction mixture pHor, more conveniently, by using an automatic titrator as a pH stat. Acombination of partial buffering capacity and base addition can also beused for process control.

When base addition is employed to neutralize gluconic acid releasedduring a ketoreductase-catalyzed reduction reaction, the progress of theconversion may be monitored by the amount of base added to maintain thepH. Typically, bases added to unbuffered or partially buffered reactionmixtures over the course of the reduction are added in aqueoussolutions.

In some embodiments, the co-factor regenerating system can comprises aformate dehydrogenase. The terms “formate dehydrogenase” and “FDH” areused interchangeably herein to refer to an NAD⁺ or NADP⁺-dependentenzyme that catalyzes the conversion of formate and NAD⁺ or NADP⁺ tocarbon dioxide and NADH or NADPH, respectively. Formate dehydrogenasesthat are suitable for use as cofactor regenerating systems in theketoreductase-catalyzed reduction reactions described herein includeboth naturally occurring formate dehydrogenases, as well asnon-naturally occurring formate dehydrogenases. Formate dehydrogenasesinclude those corresponding to SEQ ID NOS: 70 (Pseudomonas sp.) and 72(Candida boidinii) of PCT publication WO 2005/018579, which are encodedby polynucleotide sequences corresponding to SEQ ID NOS: 69 and 71,respectively, of PCT publication 2005/018579, the disclosures of whichare incorporated herein by reference. Formate dehydrogenases employed inthe methods described herein, whether naturally occurring ornon-naturally occurring, may exhibit an activity of at least about 1μmol/min/mg, sometimes at least about 10 μmol/min/mg, or at least about10² μmol/min/mg, up to about 10³ μmol/min/mg or higher, and can bereadily screened for activity in the assay described in Example 4 of PCTpublication WO 2005/018579.

As used herein, the term “formate” refers to formate anion (HCO₂ ⁻),formic acid (HCO₂H), and mixtures thereof. Formate may be provided inthe form of a salt, typically an alkali or ammonium salt (for example,HCO₂Na, KHCO₂NH₄, and the like), in the form of formic acid, typicallyaqueous formic acid, or mixtures thereof. Formic acid is a moderateacid. In aqueous solutions within several pH units of its pKa (pKa=3.7in water) formate is present as both HCO₂ and HCO₂H in equilibriumconcentrations. At pH values above about pH 4, formate is predominantlypresent as HCO₂. When formate is provided as formic acid, the reactionmixture is typically buffered or made less acidic by adding a base toprovide the desired pH, typically of about pH 5 or above. Suitable basesfor neutralization of formic acid include, but are not limited to,organic bases, for example amines, alkoxides and the like, and inorganicbases, for example, hydroxide salts (e.g., NaOH), carbonate salts (e.g.,NaHCO₃), bicarbonate salts (e.g., K₂CO₃), basic phosphate salts (e.g.,K₂HPO₄, Na₃PO₄), and the like.

For pH values above about pH 5, at which formate is predominantlypresent as HCO₂ ⁻, Equation (4) below, describes the formatedehydrogenase-catalyzed reduction of NAD⁺ or NADP⁺ by formate.

When formate and formate dehydrogenase are employed as the cofactorregeneration system, the pH of the reaction mixture may be maintained atthe desired level by standard buffering techniques, wherein the bufferreleases protons up to the buffering capacity provided, or by theaddition of an acid concurrent with the course of the conversion.Suitable acids to add during the course of the reaction to maintain thepH include organic acids, for example carboxylic acids, sulfonic acids,phosphonic acids, and the like, mineral acids, for example hydrohalicacids (such as hydrochloric acid), sulfuric acid, phosphoric acid, andthe like, acidic salts, for example dihydrogenphosphate salts (e.g.,KH₂PO₄), bisulfate salts (e.g., NaHSO₄) and the like. Some embodimentsutilize formic acid, whereby both the formate concentration and the pHof the solution are maintained.

When acid addition is employed to maintain the pH during a reductionreaction using the formate/formate dehydrogenase cofactor regenerationsystem, the progress of the conversion may be monitored by the amount ofacid added to maintain the pH. Typically, acids added to unbuffered orpartially buffered reaction mixtures over the course of conversion areadded in aqueous solutions. 101861 The terms “secondary alcoholdehydrogenase” and “sADH” are used interchangeably herein to refer to anNAD⁺ or NADP⁺-dependent enzyme that catalyzes the conversion of asecondary alcohol and NAD⁺ or NADP⁺ to a ketone and NADH or NADPH,respectively. Equation (5), below, describes the reduction of NAD⁺ orNADP⁺ by a secondary alcohol, illustrated by isopropanol.

Secondary alcohol dehydrogenases that are suitable for use as cofactorregenerating systems in the ketoreductase-catalyzed reduction reactionsdescribed herein include both naturally occurring secondary alcoholdehydrogenases, as well as non-naturally occurring secondary alcoholdehydrogenases. Naturally occurring secondary alcohol dehydrogenasesinclude known alcohol dehydrogenases from, Thermoanerobium brockii,Rhodococcus etythropolis, Lactobacillus kefir, and Lactobacillus brevis,and non-naturally occurring secondary alcohol dehydrogenases includeengineered alcohol dehydrogenases derived therefrom. Secondary alcoholdehydrogenases employed in the methods described herein, whethernaturally occurring or non-naturally occurring, may exhibit an activityof at least about 1 μmol/min/mg, sometimes at least about 10μmol/min/mg, or at least about 10² gmol/min/mg, up to about 10³gmol/min/mg or higher.

Suitable secondary alcohols include lower secondary alkanols andaryl-alkyl carbinols. Examples of lower secondary alcohols includeisopropanol, 2-butanol, 3-methyl-2-butanol, 2-pentanol, 3-pentanol,3,3-dimethyl-2-butanol, and the like. In one embodiment the secondaryalcohol is isopropanol. Suitable aryl-akyl carbinols includeunsubstituted and substituted 1-arylethanols.

When a secondary alcohol and secondary alcohol dehydrogenase areemployed as the cofactor regeneration system, the resulting NAD or NADPis reduced by the coupled oxidation of the secondary alcohol to theketone by the secondary alcohol dehydrogenase. Some engineeredketoreductases also have activity to dehydrogenate a secondary alcoholreductant. In some embodiments using secondary alcohol as reductant, theengineered ketoreductase and the secondary alcohol dehydrogenase are thesame enzyme.

In carrying out embodiments of the ketoreductase-catalyzed reductionreactions described herein employing a cofactor regeneration system,either the oxidized or reduced form of the cofactor may be providedinitially. As described above, the cofactor regeneration system convertsoxidized cofactor to its reduced form, which is then utilized in thereduction of the ketoreductase substrate.

In some embodiments, cofactor regeneration systems are not used. Forreduction reactions carried out without the use of a cofactorregenerating systems, the cofactor is added to the reaction mixture inreduced form.

In some embodiments, when the process is carried out using whole cellsof the host organism, the whole cell may natively provide the cofactor.Alternatively or in combination, the cell may natively or recombinantlyprovide the glucose dehydrogenase.

In carrying out the stereoselective reduction reactions describedherein, the engineered ketoreductase enzyme, and any enzymes comprisingthe optional cofactor regeneration system, may be added to the reactionmixture in the form of the purified enzymes, whole cells transformedwith gene(s) encoding the enzymes, and/or cell extracts and/or lysatesof such cells. The gene(s) encoding the engineered ketoreductase enzymeand the optional cofactor regeneration enzymes can be transformed intohost cells separately or together into the same host cell. For example,in some embodiments one set of host cells can be transformed withgene(s) encoding the engineered ketoreductase enzyme and another set canbe transformed with gene(s) encoding the cofactor regeneration enzymes.Both sets of transformed cells can be utilized together in the reactionmixture in the form of whole cells, or in the form of lysates orextracts derived therefrom. In other embodiments, a host cell can betransformed with gene(s) encoding both the engineered ketoreductaseenzyme and the cofactor regeneration enzymes.

Whole cells transformed with gene(s) encoding the engineeredketoreductase enzyme and/or the optional cofactor regeneration enzymes,or cell extracts and/or lysates thereof, may be employed in a variety ofdifferent forms, including solid (e.g., lyophilized, spray-dried, andthe like) or semisolid (e.g., a crude paste).

The cell extracts or cell lysates may be partially purified byprecipitation (ammonium sulfate, polyethyleneimine, heat treatment orthe like, followed by a desalting procedure prior to lyophilization(e.g., ultrafiltration, dialysis, and the like). Any of the cellpreparations may be stabilized by crosslinking using known crosslinkingagents, such as, for example, glutaraldehyde or immobilization to asolid phase (e.g., Eupergit C, and the like).

The solid reactants (e.g., enzyme, salts, etc.) may be provided to thereaction in a variety of different forms, including powder (e.g.,lyophilized, spray dried, and the like), solution, emulsion, suspension,and the like. The reactants can be readily lyophilized or spray driedusing methods and equipment that are known to those having ordinaryskill in the art. For example, the protein solution can be frozen at−80° C. in small aliquots, then added to a prechilled lyophilizationchamber, followed by the application of a vacuum. After the removal ofwater from the samples, the temperature is typically raised to 4° C. fortwo hours before release of the vacuum and retrieval of the lyophilizedsamples.

The quantities of reactants used in the reduction reaction willgenerally vary depending on the quantities of product desired, andconcomitantly the amount of ketoreductase substrate employed. Thefollowing guidelines can be used to determine the amounts ofketoreductase, cofactor, and optional cofactor regeneration system touse. Generally, keto substrates can be employed at a concentration ofabout 20 to 300 grams/liter using from about 50 mg to about 5 g ofketoreductase and about 10 mg to about 150 mg of cofactor. Those havingordinary skill in the art will readily understand how to vary thesequantities to tailor them to the desired level of productivity and scaleof production. Appropriate quantities of optional cofactor regenerationsystem may be readily determined by routine experimentation based on theamount of cofactor and/or ketoreductase utilized. In general, thereductant (e.g., glucose, formate, isopropanol) is utilized at levelsabove the equimolar level of ketoreductase substrate to achieveessentially complete or near complete conversion of the ketoreductasesubstrate.

The order of addition of reactants is not critical. The reactants may beadded together at the same time to a solvent (e.g., monophasic solvent,biphasic aqueous co-solvent system, and the like), or alternatively,some of the reactants may be added separately, and some together atdifferent time points. For example, the cofactor regeneration system,cofactor, ketoreductase, and ketoreductase substrate may be added firstto the solvent.

For improved mixing efficiency when an aqueous co-solvent system isused, the cofactor regeneration system, ketoreductase, and cofactor maybe added and mixed into the aqueous phase first. The organic phase maythen be added and mixed in, followed by addition of the ketoreductasesubstrate. Alternatively, the ketoreductase substrate may be premixed inthe organic phase, prior to addition to the aqueous phase

Suitable conditions for carrying out the ketoreductase-catalyzedreduction reactions described herein include a wide variety ofconditions which can be readily optimized by routine experimentationthat includes, but is not limited to, contacting the engineeredketoreductase enzyme and substrate at an experimental pH and temperatureand detecting product, for example, using the methods described in theExamples provided herein.

The ketoreductase catalyzed reduction is typically carried out at atemperature in the range of from about 15° C. to about 75° C. For someembodiments, the reaction is carried out at a temperature in the rangeof from about 20° C. to about 55° C. In still other embodiments, it iscarried out at a temperature in the range of from about 20° C. to about45° C. The reaction may also be carried out under ambient conditions.

The reduction reaction is generally allowed to proceed until essentiallycomplete, or near complete, reduction of substrate is obtained.Reduction of substrate to product can be monitored using known methodsby detecting substrate and/or product. Suitable methods include gaschromatography, HPLC, and the like. Conversion yields of the alcoholreduction product generated in the reaction mixture are generallygreater than about 50%, may also be greater than about 60%, may also begreater than about 70%, may also be greater than about 80%, may also begreater than 90%, and are often greater than about 97%.

EXPERIMENTAL

Various features and embodiments of the disclosure are illustrated inthe following representative examples, which are intended to beillustrative, and not limiting.

In the experimental disclosure below, the following abbreviations apply:ppm (parts per million); M (molar); mM (millimolar), uM and μM(micromolar); nM (nanomolar); mol (moles); gm and g (gram); mg(milligrams); ug and ng (micrograms); L and 1 (liter); ml and mL(milliliter); cm (centimeters); mm (millimeters); um and μm(micrometers); sec. (seconds); min(s) (minute(s)); h(s) and hr(s)(hour(s)); U (units); MW (molecular weight); rpm (rotations per minute);° C. (degrees Centigrade); RT (room temperature); CDS (coding sequence);DNA (deoxyribonucleic acid); RNA (ribonucleic acid); HPLC (highperformance liquid chromatography); FIOPC (fold improvement overpositive control); HTP (high throughput); LB (Luria broth);Sigma-Aldrich (Sigma-Aldrich, St. Louis, Mo.); Millipore (Millipore,Corp., Billerica Mass.); Difco (Difco Laboratories, BD DiagnosticSystems, Detroit, Mich.); Daicel (Daicel, West Chester, Pa.); Genetix(Genetix USA, Inc., Beaverton, Oreg.); Molecular Devices (MolecularDevices, LLC, Sunnyvale, Calif.); Applied Biosystems (AppliedBiosystems, part of Life Technologies, Corp., Grand Island, N.Y.),Agilent (Agilent Technologies, Inc., Santa Clara, Calif.); ThermoScientific (part of Thermo Fisher Scientific, Waltham, Mass.); Corning(Corning, Inc., Palo Alto, Calif.); and Bio-Rad (Bio-Rad Laboratories,Hercules, Calif.).

In some experiments, samples were analyzed by reversed phase highperformance liquid chromatography (HPLC) to quantify residual substratesand products. Reactions (10 μL) were analyzed across a ChiralPak AS-3Rcolumn (100×4.6 mm, 3 μm particle size) (Daicel, West Chester, Pa.) at25-50° C. eluting with 30-35% acetonitrile, 65-70% water, and 0.1%phosphoric acid at 1-1.5 ml/min. Reactants and products were quantifiedby their UV detection at 210 or 254 nm. In one embodiment, samples wereanalyzed at 30° C. with 35% acetonitrile, 65% water and 0.1% phosphoricacid at 1 ml/min. A racemic mixture of substrate eluted at 8.0 mM,product 2a eluted at 4.2 mM, 2c eluted at 4.6 min, 2b eluted at 5.3 mM,and 2d eluted at 6.1 mM.

Example 1 Ketoreductase Gene Construction and Expression Vectors

The wild-type L. kifer ketoreductase (KRED) encoding gene was designedfor expression in E. coli based on the reported amino acid sequence ofthe ketoreductase and a codon optimization algorithm as described inExample 1 of WO2008/042876, incorporated herein by reference. The genewas synthesized using oligonucleotides composed of 42 nucleotides andcloned into expression vector pCK11 0900 (See, FIG. 3 of US Pat. Appln.Publn. No. 2006/0195947, herein incorporated by reference) under thecontrol of a lac promoter. The expression vector also contained the P15aorigin of replication and the chloramphenicol resistance gene. Theactivity of the wild-type ketoreductase was confirmed as described inWO2008/042876. Polynucleotides encoding engineered ketoreductases of thepresent disclosure were likewise cloned into vector pCK11 0900 forexpression in E. coli W311 0. Directed evolution of the KRED gene wascarried out by first selecting the parent gene (i.e., SEQ ID NOS: 4, 9,25, 41) followed by library construction of variant genes in whichpositions associated with certain structural features were subjected tomutagenesis. These libraries were then plated, grown-up, and screenedusing HTP assays as described in Examples 2 through 7.

Example 2 Production and Analysis of Engineered KRED Polypeptides

Plasmid libraries obtained through directed evolution and containingevolved ketoreductase genes were transformed into E. coli W3110 andplaced on Luria-Bertani (LB) agar medium containing 1% glucose and 30μg/ml chloramphenicol (CAM). After incubation for at least 16 h at 30°C., colonies were picked using a Q-bot® robotic colony picker (Genetix)into a 96-well shallow well microtiter plate containing 200 μL of LB, 1%glucose, and 30 μg/ml CAM. Cells were grown 18-20 h at 30° C., withshaking at 200 rpm. Twenty μL of this culture was then transferred to360 μL of Terrific Broth (TB) and 30 μg/ml CAM. After incubation of deepwell plates at 30° C. with shaking at 250 rpm for 2.5 h (OD₆₀₀ 0.6-0.8),recombinant gene expression was induced by isopropyl thioglycoside(IPTG) to a final concentration of 1 mM. The plates were then incubatedat 30° C. with shaking at 250 rpm for 18-21 h.

Cell cultures were pelleted at 3500×g for 20 mM, and their supernatantswere discarded. Cell pellets were lysed in 300 μL of 20 mM Tris, 1 mMMgCl₂ pH 7.5 with 1 g/L lysozyme and 0.5 g/L polymixin B sulfate byshaking at RT for 2 h. Samples were centrifuged at 3500×g for 20 mM toclarify cellular debris, and the supernatant was used to carry out thetransformations described in Examples 3 through 7.

Example 3 KRED Variants of SEQ ID NO:4

E. coli KRED variants were generated as described in Example 1. Toanalyze the selectivity of the variants, 25 μL supernatant produced asdescribed in Example 2 was added to a mixture of 50 μL substrate (4 g/L)in isopropanol and 25 μL of 4 g/L NADP in 400 mM sodium phosphate pH9.0. Reactions were incubated at 50° C. for 1 h, and quenched viaaddition of 100 μL CH₃CN. The quenched mixture (100 μL) was added to 50μL of 0.3% phosphoric acid, mixed briefly. Reaction samples (10 μL) wereanalyzed by reversed phase HPLC to quantify residual substrates andproducts as described above. Significantly improved variants areprovided in Table 4.1, below.

TABLE 4.1 Variants With Improved 2a: 2c Selectivity Compared to SEQ IDNO: 4 Amino acid Substitutions Relative Selectivity SEQ ID NO: to SEQ IDNO: 4 Improvement 6 F145W; D150P ++ 8 F249K + 10 F249R + 12 P94G;F249K + 14 P94G; P190W + 16 V144G ++ 18 V144G; F145W ++ Key for Table4.1 +++ >2.5 ++ >1.8 and <2.5 + >1.3 and <1.8

Example 4 KRED Variants of SEQ ID NO:9

E. coli KRED variants were generated as described in Example 1. Toanalyze the selectivity of the variants, 25 μL supernatant produced asdescribed in Example 2 supernatant was added to 180 μL of 20 mM Tris, 1mM MgCl₂ pH 7.5. The diluted supernatant (25 μL) was added to a mixtureof 50 μL substrate (2 g/L) in isopropanol and 25 μL of 4 g/L NADP in 400mM sodium phosphate pH 9.0. Reactions were incubated at 50° C. for 1 h,and quenched via addition of 100 μL CH₃CN. The quenched mixture (100 μL)was added to 50 μL of 0.3% phosphoric acid, mixed briefly. Reactionsamples (10 μL) were analyzed by reversed phase HPLC to quantifyresidual substrates and products as described above. Significantlyimproved variants are provided in Table 5.1, below.

TABLE 5.1 Variants With Improved 2a: 2c Selectivity Compared to SEQ IDNO: 10 Amino acid Substitutions Relative Selectivity SEQ ID NO: to SEQID NO: 10 Improvement 20 D150W; F206Y; K211I + 22 F145W; D150F; T153C; +P190A; D198G 24 F145W; D150W; T153C; + F206Y; K211H 26 F145W; D150W;T153C; + K211E 28 F145W; P190A + 30 F145W; T153C; K211I + 32 F206Y;K211I + 34 P190A; D198G; F206Y; + K211H 36 P190A; F206Y + 38 T153C;P190A; F206Y; + K211S Key for Table 5.1 +++ >2.5 ++ >1.8 and <2.5 + >1.3and <1.8

Example 5 KRED Variants of SEQ ID NO:25

E. coli KRED variants were generated as described in Example 1. Toanalyze the selectivity of the variants, 25 μL supernatant produced asdescribed in Example 2 was added was added to a mixture of 50 μLsubstrate (1 g/L) in isopropanol and 25 μL of 0.4 g/L NADP in 400 mMsodium phosphate pH 9.0. Reactions were incubated at 50° C. for 2.5 h,and quenched via addition of 100 μL CH₃CN. The quenched mixture (100 μL)was added to 50 μL of 0.3% phosphoric acid, mixed briefly. Reactionsamples (10 μL) were analyzed by reversed phase HPLC to quantifyresidual substrates and products as described above. Significantlyimproved variants are provided in Table 6.1, below.

TABLE 6.1 Variants With Improved 2a: 2c 1 Selectivity Compared to SEQ IDNO: 26 Amino acid Substitutions Relative SEQ ID NO: to SEQ ID NO: 26Selectivity Improvement 40 C153T; R209M ++ 42 P94M; C153T; Y199E +++ 44T212E ++ 46 C153T; G201N; T212G ++ 48 T212H ++ 50 P190Q ++ 52 C153H;A175T ++ 54 T212R ++ 56 R209I + 58 R209F ++ 60 P213M ++ 62 P213D ++ 64R209E ++ 66 C153T; A202H + 68 R209W + 70 P213R ++ 72 P213K + 74 P213E +76 C153T; S207D + 78 A202H + 80 P213T + 82 R209T + 84 P94Q + 86 A202F +88 C153T; P213H + 90 R209V + 92 C153T; A202Y + 94 C153T; M196N + 96P213N + 98 E211P + 100 S207D + 102 P213G + 104 C153H + 106 A202Y + 108R209G + 110 T210L + 112 C153T; S207G + 114 C153T; E203L + 116 C153T;T210P + 118 T212G + 120 M196K + 122 I217N + 124 I147L; A202Y + 126S207Y + 128 A202R + 130 R209Y + 132 C153T; F206H + 134 P190L; M196I +136 C153T; F206W + 138 C153T; E203W + Key for Table 6.1 +++ >2.5 ++ >1.8and <2.5 + >1.3 and <1.8

Example 6 KRED Variants of SEQ ID NO:41

E. coli KRED variants were generated as described in Example 1. Toanalyze the selectivity of the variants, 30 μL supernatant produced asdescribed in example 2 was added to a mixture of 75 μL substrate (20g/L) in isopropanol and 45 μL of 0.33 g/L NADP in 333 mM sodiumphosphate pH 8.0. Reactions were incubated at 30° C. for 1 h, andquenched via addition of 100 μL CH₃CN. The quenched mixture (100 μL) wasadded to 50 μL of 0.3% phosphoric acid, mixed briefly. Reaction samples(10_(N)L) were analyzed by reversed phase HPLC to quantify residualsubstrates and products as described above. Significantly improvedvariants are provided in Table 7.1, below.

TABLE 7.1 Samples With Improved 2a: 2c Selectivity Compared to SEQ IDNO: 42 Amino acid Substitutions Relative SEQ ID NO: to SEQ ID NO: 42Selectivity Improvement 140 P190L; M196N; I217H + 142 T102D; W150D + 144P190T; T212N + 146 T212N; Q252W + 148 R209G ++ 150 L110E ++ 152 M196N ++154 T212A; T250G ++ 156 G149S; W150D; E203W ++ 158 D197Y; Q252W ++ 160T212A; Q252W ++ 162 N114G; M196H ++ 164 W150D; E203G ++ 166 M196Q ++ 168M196H; T212A ++ 170 T212P ++ 172 M94Q; T212H ++ 174 G149S; W150D ++ 176T212N; T250G ++ 178 M196H; D197Y ++ 180 D197Y; T250G; Q252W ++ 182T212A; T250G; Q252W ++ 184 M205T; S207V; T212A; ++ Q252W 186 M94Q; P213G++ 188 L110E; T212V ++ 190 W150D; A158V +++ 192 W150D; A158V; D198S +++194 T153P; T212P +++ 196 W150D; D198S; E203W +++ 198 P213M +++ 200W150D; D198S; E203L +++ 202 W150D +++ 204 T212V; P213N +++ 206 T212R +++208 P213E +++ 210 M196H +++ 212 P213T +++ 214 P213G +++ 216 T212E; P213G+++ 218 L110W; P213N +++ 220 P213N +++ 222 T212G; P213N +++ 224 T212H;P213K +++ 226 P213K +++ 228 T212R; P213G +++ 230 A68V + 232 G135K 234D233T 236 D233Q 238 D173L; R249G +++ Key for Table 7.2 +++ >2.5 ++ >1.8and <2.5 + >1.3 and <1.8

Example 7 Production of Engineered Polypeptides (Shake Flask ScaleProduction of KREDs) and Performance Validation

Plasmid libraries obtained through directed evolution and containingevolved ketoreductase genes were transformed into E. coli W3110 andplaced on Luria-Bertani (LB) agar medium containing 1% glucose and 30μg/ml chloramphenicol (CAM). After incubation for at least 16 h at 30°C., colonies were picked into 50 mL of LB, 1% glucose, and 30 μg/ml CAM.Cells were grown 18-20 h at 30° C., with shaking at 250 rpm. Thisculture was then transferred into Terrific Broth (TB) and 30 μg/ml CAMat a final OD₆₀₀ of 0.2 and a final volume of 250 mL. After incubationof the flasks at 30° C. with shaking at 250 rpm for 2.5 h (OD₆₀₀0.6-0.8), recombinant gene expression was induced by isopropylthioglycoside (IPTG) to a final concentration of 1 mM. The flask wasthen incubated at 30° C. with shaking at 250 rpm for 18-21 h. Cells werepelleted at 3500×g for 20 mM, and the supernatant was discarded. Thecell pellet was washed 3× in 30 mL ice cold 50 mM sodium phosphate pH7.5, resuspended in 12 ml of the same buffer, and lysed using a celldisruptor at 18-20 kpsi. Lysates were clarified at 10000×g for 30 mM,and clarified supernatants were lyophilized to an off white powder.

To evaluate the final compound under process like conditions, 500 μL ofisopropanol was added to 50 mg of substrate followed by 500 μL of 200 mMsodium phosphate pH 8.0, 0.2 g/L NADP, and 1 g/L of lyophilized powderof SEQ ID NO:238 generated as described above. Reactions were heated to50° C. with shaking at 950 rpm for 24 h resulting in 95% conversion ofsubstrate to product and a 92% diastereomeric excess of 2a to 2c.Reaction samples were analyzed by reversed phase HPLC to quantifyresidual substrates and products as described above.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

What is claimed is:
 1. An engineered polynucleotide encoding anengineered polypeptide comprising an amino acid sequence with at least90% sequence identity to SEQ ID NO:4 and an arginine or lysine atposition X249 as compared to SEQ ID NO:4, and wherein said polypeptidehas ketoreductase activity with at least 2-fold greater selectivity tothe chiral compound 2a relative to chiral compound 2c as compared to SEQID NO:4.
 2. The engineered polynucleotide encoding the engineeredpolypeptide of claim 1, wherein said amino acid sequence furthercomprises at least one of the following: the residue corresponding toX68 is a non-polar, or aliphatic residue; the residue corresponding toX102 is an acidic residue; the residue corresponding to X110 is anacidic, or aromatic residue; the residue corresponding to X114 is anon-polar residue; the residue corresponding to X135 is a basic residue;the residue corresponding to X144 is a non-polar, or aromatic residue;the residue corresponding to X147 is a non-polar, or aliphatic residue;the residue corresponding to X149 is a polar residue; the residuecorresponding to X153 is a polar, or aromatic residue; the residuecorresponding to X158 is a non-polar, or aliphatic residue; the residuecorresponding to X175 is a polar residue; the residue corresponding toX190 is an aromatic, aliphatic, non-polar, or polar residue; the residuecorresponding to X196 is an aromatic, polar, basic, non-polar, oraliphatic residue; the residue corresponding to X197 is an aromaticresidue; the residue corresponding to X198 is a non-polar, polarresidue; the residue corresponding to X201 is a polar residue; theresidue corresponding to X202 is an aromatic, or basic residue; theresidue corresponding to X203 is an aromatic, non-polar, or aliphaticresidue; the residue corresponding to X205 is a polar residue; theresidue corresponding to X206 is an aromatic residue; the residuecorresponding to X207 is an aromatic, acidic, non-polar, or aliphaticresidue; the residue corresponding to X209 is an aromatic, acidic,non-polar, polar, or aliphatic residue; the residue corresponding toX210 is a non-polar, or aliphatic residue; the residue corresponding toX212 is an aromatic, basic, non-polar, aliphatic, or acidic residue; theresidue corresponding to X213 is an aromatic, basic, acidic, polar,non-polar, or aliphatic residue; the residue corresponding to X217 is apolar, or aromatic residue; the residue corresponding to X233 is a polarresidue; the residue corresponding to X250 is a non-polar residue; andthe residue corresponding to X252 is an aromatic residue.
 3. Theengineered polynucleotide encoding the engineered polypeptide of claim1, further comprising at least one substitution selected from X68V,X94G/Q, X102D, X110E/W, X114G, X135K, X144G/W, X147L, X149S,X150D/F/P/W, X153H/P/T/V/C, X158V, X175T, X190A/L/Q/T/W, X196H/I/K/N/Q,X1961, X196K/N/Q, X197Y, X198G/S, X201N, X202F/H/R/Y, X203G/L/W, X205T,X206H/W/Y, X207D/G/V/Y, X209E/F/G/I/M/T/V/W, X210L/P, X211H/I/P/S,X212A/E/G/H/N/P/R/V, X213D/E/G/H/K/M/N/R, X217H/N, X233Q/T, X250G, andX252W.
 4. The engineered polynucleotide encoding the engineeredpolypeptide of claim 1, further comprising a substitution at positionX190.
 5. The engineered polynucleotide encoding the engineeredpolypeptide of claim 4, comprising a substitution at position X190,wherein the substitution is selected from P, A, T, or Q.
 6. Theengineered polynucleotide encoding the engineered polypeptide claim 1,wherein the stereoselective activity of the polypeptide is increased atleast 2-fold as compared to the corresponding activity of the referencepolypeptide of SEQ ID NOS: 4, 10, 26, and/or
 42. 7. The engineeredpolynucleotide of claim 1, in which the polynucleotide comprises anucleic acid sequence selected from the group consisting of theodd-numbered sequence identifiers of SEQ ID NOS:5-237.
 8. An expressionvector comprising the polynucleotide of claim
 1. 9. A host cellcomprising the polynucleotide of claim
 1. 10. A host cell comprising theexpression vector of claim
 8. 11. The host cell of claim 9, wherein saidhost cell is E. coli.
 12. The host cell of claim 10, wherein said hostcell is E. coli.